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Biomedical subjects

D O DeFouw

Publications and source records attributed to D O DeFouw.

At least 37 records · Page 2Linked to original sources
PubMed1995

Differential lectin binding to microvascular endothelial glycoconjugates during normal angiogenesis in the chick chorioallantoic membrane.

The ontogenesis of endothelial glycoconjugate expression during normal angiogenesis and microvascular development remains unknown. Using intravital fluorescent microscopy, we studied temporal and spatial lectin binding to carbohydrate moieties of luminal microvascular endothelia of the chick embryo chorioallantoic membrane (CAM) during Days 4.5 to 6.0 of the 21-day incubation. We used a battery of eight FITC-lectins (100-200 micrograms/ml). Fluorescent images from precapillary, capillary, and postcapillary segments of the lectin-perfused microvascular units were analyzed by image analysis software to quantitate differences in lectin binding. Results served to indicate a significant decrease in lectin binding to terminal N-acetyl glucosamine, N-acetyl galactosamine, and the N-acetyl galactosamine disaccharide in the glycocalyx of pre- and postcapillary vessels from Day 4.5 to Day 5.0. Lectin binding to N-acetyl glucosamine and N-acetyl galactosamine subsequently increased at Days 5.0 and 6.0. In the capillaries, lectin binding to endothelial galactose, fucose, and sialic acid increased significantly from Day 5.5 to Day 6.0. That these temporal changes in lectin binding to endothelial luminal glycoconjugates coincide with concomitant changes in CAM microvascular permeability (Rizzo et al., in press) serves to suggest a possible association between expression of endothelial glycoconjugates and the ontogeny of microvascular perm-selectivity during normal angiogenesis.

Allantois↗
PubMed1995

Differentiation of the microvascular endothelium during early angiogenesis and respiratory onset in the chick chorioallantoic membrane.

The present study served to determine the extent of microvascular endothelial differentiation during early stages of morphogenesis (days 4.5-5.5 of the 21-day incubation) in the chick chorioallantoic membrane (CAM). CAM's, which serve as the embryonic lung, were prepared for intravital injections of a graded series of FITC-dextrans and subsequent ultrastructural morphometric analyses of the microvascular units. The precapillary, capillary, and postcapillary microvascular segments presented a continuous endothelium that was substantially thicker than that of adult lung endothelia (DeFouw, 1988). Further, plasmalemmal vesicles were uniformly sparse, while endothelial vacuoles, of variable diameters, were present continuously in the proliferating microvascular units. Average widths and depths of the interendothelial clefts were uniform and suggested complete structural differentiation from the onset of CAM morphogenesis. Based on our recent estimates of CAM microvascular permeability coefficients (Rizzo et al., 1995), the observed endothelial ultrastructure was associated with microvascular selectivity comparable to that of adult pulmonary microvessels (Lanken et al., 1985). Therefore, despite incomplete ultrastructural differentiation of the early CAM microvascular endothelium, these angiogenic microvessels presented adult-like barrier properties. Further they were less permeable than (Wu et al., 1993; Yuan et al., 1993) and ultrastructurally distinct from (Kohn et al., 1992) certain tumorigenic microvessels. Thus, angiogenesis is likely not a routinely homogeneous process, and CAM microvascular permeability characteristics may be teleologically significant.

Allantois↗
PubMed1993

The microvascular unit of the 6-day chick chorioallantoic membrane: a fluorescent confocal microscopic and ultrastructural morphometric analysis of endothelial permselectivity.

The chorioallantoic membrane (CAM) of the chick embryo provides a unique model for investigating endothelial permselectivity during normal angiogenesis. Chick embryos were incubated using established shell-less culture techniques for intravital and ultrastructural observations at Day 6 of the normal 21-day gestation. Morphometric analyses of the precapillary, capillary, and postcapillary microvascular segments served to demonstrate a continuous endothelium of uniform cytoplasmic thickness and few plasmalemmal vesicles. Average widths and depths of the endothelial junctional clefts were also homogeneous, except the significantly longer junctional depths of the second-order postcapillaries. The frequency of endothelial junctions per unit length of segmental luminal surface, like the vesicle densities, were uniformly less than those reported previously for adult continuous endothelia. Qualitative observations of systemically microinjected FITC-dextrans (40, 70, and 150 KDa), using real-time fluorescent confocal microscopy, failed to identify substantial interstitial accumulation of any dextrans during 10-min perfusion periods. Ultrastructural examination of the same dextran probes, on the other hand, served to detect small, sporadic interstitial foci of the dextran tracers. Such foci created a wide scattering of particle counts in the extravascular space, and extravascular accumulation was not sufficient to elicit an observable fluorescent signal. These results are consistent with the interpretation that during early stages of the normal angiogenic process, macromolecules > or = 40,000 MW can traverse the CAM segmental microvascular endothelium. The extent of extravasation, however, was less than that reported previously during pathologic angiogenesis associated with wound healing or tumor growth.

Allantois↗
PubMed1993

Effect of extracellular fluid volume expansion on avian lung fluid balance.

We examined the effects of acute expansion of extracellular fluid volume (ECFV) on lung fluid balance and the ultrastructure of the pulmonary air-blood barrier in chickens (Gallus domesticus). We compared changes in extravascular lung water content (EVLW) to the sum of mean pulmonary capillary blood pressure and plasma protein osmotic pressure (tau c), as a measure of net intravascular filtration pressure (NIFP), produced by graded infusion of avian Ringer's solution. NIFP increased with each volume load largely as a result of decreased tau c resulting in progressive increase in EVLW. Progressive interstitial edema occurred with fluid accumulation restricted to the inter air capillary septa, sparing the gas exchanging regions of the air-blood barrier. This was associated with increased thickness of the septa and increased pulmonary capillary endothelial vesiculation. The effect of increased ECFV on pulmonary hemodynamics and lung fluid balance in Gallus is similar to that in mammals.

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PubMed1993

Macromolecular selectivity of chick chorioallantoic membrane microvessels during normal angiogenesis and endothelial differentiation.

Progressive angiogenesis and endothelial differentiation in the chick chorioallantoic membrane (CAM) serve to accommodate oxygen demands of the growing embryo. The present study evaluated CAM microvascular endothelial permselectivity during the most rapid phase of angiogenesis (day 10) and after initiation of endothelial cytodifferentiation (day 14). Chick embryos were incubated using established shell-less culture techniques for intravital and ultrastructural observations. Systemic microinjections of FITC-dextrans (40, 70 and 150 KDa) provided an index of endothelial permselectivity after 2.5 min and 10 min perfusions. Ultrastructural examinations of the same dextran probes served to detect small, intermittent foci within the perivascular interstitium. Although minor variations of dextran particle distributions around specific segments of the microcirculation were observed ultrastructurally, perivascular accumulation was not sufficient to elicit a detectable fluorescent signal. Thus, substantial accumulation of the graded-dextran series in the perivascular interstitium was not detected. Morphometric analyses of the precapillary, capillary, and postcapillary microvascular segments served to demonstrate a continuous endothelium which displayed cytoplasmic attenuation at day 14. Plasmalemmal vesicles were few and uniform within the microvascular units at day 10. A three-fold increase in vesicle densities characterized the precapillary endothelia at day 14. Average widths of the endothelial junctional clefts were homogeneous within the segmental microvascular endothelia at both days 10 and 14. Junctional cleft lengths were also homogeneous, except the significantly longer capillary endothelial clefts observed at day 10. These results are consistent with the concept that, despite certain differences in segmental vesicle densities and junctional cleft lengths, neovascularization of the CAM is achieved without excessive macromolecular efflux across the microvascular endothelia.

Allantois↗
PubMed1989

Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesis.

The microcirculation within the chorioallantoic membrane (CAM) of the chick is particularly well suited for in vivo observation and has been used extensively as an assay to detect angiogenic activity. Although progressive chronological expansion of the CAM capillary network occurs normally during embryogenesis, descriptions of the branching patterns of CAM pre- and postcapillary microvessels during embryonic development have not been recorded. In the present study chick embryos were incubated, using an established shell-less culture technique, and observed in vivo at Days 6, 10, and 14 of embryonic development. Morphometric analyses of photomicrographs of CAM microvessels were based upon the centripetal ordering method of microvascular mapping of the first three orders of pre- and postcapillary microvessels with the capillaries serving as the initial point of reference. For both pre- and postcapillary vessels, the number of first-order vessels exceeded the number of second-order vessels which, in turn, outnumbered third-order vessels during each observation period. First- and second-order vessels progressively increased in number from Day 6 to Day 14; however, the number of third-order vessels remained essentially constant during this period. Further, the number of precapillary vessels was greater than postcapillary vessels in their respective orders at Days 6 and 10; however, by Day 14 the numbers were comparable. Average diameters and lengths of the third-order vessels were greater than the second-order vessels which, in turn, were greater than the first-order vessels in both the pre- and postcapillary compartments. Further, mean lengths of each of the three vessel orders in both compartments decreased progressively and by Day 14 were significantly less than at Day 6. Average diameters of each vessel order, on the other hand, remained unchanged from Day 6 to Day 14. Finally, intercapillary distances, based on measurements from fluorescent micrographs obtained after microinjections of fluorescein isothiocyanate (FITC)-dextran, were substantially less at Day 10 and 14 than at Day 6. Based on these morphometric data, the endothelial precursor responsible for continuous neoformation of first- and second-order microvessels during embryogenesis remains uncertain. Whether existing first-, second-, or third-order vessel endothelia serve as this precursor or histodifferentiation of existing capillaries enables continuous expansion of the first- and second-order microvessels remains to be tested.

Allantois↗
PubMed1988

Structural heterogeneity within the pulmonary microcirculation of the normal rat.

Extra-alveolar microvessels of the rat were described previously as nonmuscular, partially muscular, and muscular (Hislop and Reid, 1978). Based on these structural criteria, the present morphometric results present evidence that mean thickness of the endothelium was significantly greater in the muscular microvessels than in each of the other microvessel categories. The percentage of endothelial surface occupied by attenuated segments (20-30 nm maximal thickness) was greatest in the nonmuscular and partially muscular microvessels. Mean plasmalemmal vesicle diameters were consistent throughout the microvascular units. Vesicle numerical densities, however, were significantly greater in the alveolar capillary endothelium than in endothelia of the nonmuscular, partially muscular, and muscular microvessels. Endothelial vacuoles (diameters greater than 150 nm) were not observed in either the capillaries or the nonmuscular microvessels. Mean diameters and numerical densities of vacuoles observed in endothelia of the partially muscular and muscular microvessels were, however, statistically similar. Subclassification of the partially muscular and muscular microvessels by ultrastructural characteristics as arterioles or venules (Rhodin, 1978; Michel, 1982) served to identify greater numerical densities of vesicles in the endothelium of venules than that of the arterioles. As expected, mean thicknesses of the periendothelial connective tissue spaces varied directly with the relative muscularity of the respective microvessels. Based upon the morphometric data presented in this study, future investigations of pulmonary microvascular functions should include considerations of the observed regional variations of endothelial cell ultrastructure.

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PubMed1988

The influence of circulatory overload on extraalveolar microvessel endothelium of dog lung in vivo.

In an earlier study from this laboratory, morphometric evaluations of the alveolar capillary endothelium of the lungs of intact dogs were recorded after periods of sustained increases of pulmonary microvascular pressures (D. O. DeFouw, W. O. Cua, and F. P. Chinard, 1983, Microvasc. Res. 25, 56-67). In the present study ultrastructural characteristics of the extraalveolar microvessel endothelium of these dog lungs were evaluated. Small (25- to 50-microns luminal diameter) nonmuscular vessels, which adjoin the alveolar capillaries, and larger (51-200 microns) partially muscular and muscular microvessels were assessed. After increased microvascular filtration, to be expected from the increased hydrostatic pressures, fluid accumulation was found only in the connective tissue sleeves of the larger microvessels. The endothelium of these partially muscular and muscular vessels was markedly affected by fluid distension of the periendothelial interstitium. The endothelial response included the appearance of basal (abluminal) surface invaginations, de novo plasmalemmal vesicle formation, and increased numbers of cytoplasmic vacuoles. The small nonmuscular microvessels lacked both the fluid cuffs and the alterations of endothelial ultrastructure. This latter observation is consistent with the previous report from this laboratory that indicated an absence of both alveolar septal edema and increased capillary endothelial vesicle densities in these lungs (DeFouw et al., 1983). Thus, it seems likely that the conformational changes of the endothelium of the larger microvessels were related to the formation of the periendothelial fluid cuffs. The mechanisms responsible for this endothelial response have not been determined but can be explained on the basis of the testable hypothesis that they are secondary to an increase of tissue pressure associated with accumulation of tissue fluid. These changes may thus represent a secondary structural adaptation to the increased tissue pressures and may serve as a potential vesicular-vacuolar pathway across the endothelium.

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PubMed1988

The pulmonary microcirculation of the rat: differential ultrastructural responses of the endothelia to protamine sulfate.

Protamine sulfate is used clinically to reverse the anti-coagulant effects of heparin and in certain cases high protein, non-cardiogenic pulmonary edema develops. In the present study an initial stage of edema formation, namely, interstitial fluid accumulation around partially muscular extra-alveolar microvessels was observed in rats in situ after right ventricular injections of protamine. In addition, the endothelium of these microvessels displayed marked increases in plasmalemmal vesicles; however, disruption of the endothelium was not observed. Further, endothelial vesicle densities were unchanged and perivascular cuffs were not observed in either the nonmuscular extra-alveolar microvessels or the alveolar capillaries. Left ventricular injections of protamine failed to elicit the ultrastructural responses to protamine. Predosing the pulmonary microcirculation with heparin also served to prevent protamine-induced changes in the partially muscular microvessels. If it is assumed that heparin lowers the threshold for protamine-mediated responses in patients who develop edema, inhibition of protamine-induced changes by heparin predosing cannot be explained by the present data. Although evidence of increased endothelial vesiculation in the partially muscular microvessels was obtained, relative contributions of vesicles or of the junctional clefts to efflux from the pulmonary microvessels is not known. Thus, the mechanisms associated with a reduction of endothelial selectivity to macromolecular efflux after protamine administration remain to be defined.

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PubMed1987

The response of subpleural pulmonary capillary endothelium to hydrothorax in rats.

The principal focus of this study was to evaluate the hypothesis that increased interstitial fluid pressures served to stimulate de novo vesicle formation in pulmonary capillary endothelium. Direct measurements of interstitial fluid pressures within the alveolar septa pose great technical difficulty. The pleural space and subpleural capillaries are easily accessible, and thus, provide a more feasible model to test this hypothesis. After hydrostatic pressure of pleural space fluid was increased by periodic saline infusions into the pleural cavity, vesicle numerical densities were significantly increased in portions of the subpleural capillary endothelium. Those segments of the endothelium that directly apposed the interstitium of the visceral pleura displayed de novo vesicle formation. The endothelial segments located immediately adjacent to the alveolar epithelium were not affected by the elevated interstitial fluid pressures. In addition to the increased vesiculation, those same segments of the endothelium were characterized by increased attenuation of their cytoplasmic compartments. These conformational changes in the plasmalemma of portions of the subpleural capillary endothelium provide support to the tentative hypothesis, however, whether the increased numbers of vesicles contribute to a potential transendothelial transport system or expand a possible static network of membrane invaginations remains uncertain.

Animals↗
PubMed1986

Pulmonary microcirculation: differences in endothelia of subpleural and alveolar capillaries.

Portions of the endothelia in the alveolar and subpleural capillary networks appose either the adjacent alveolar epithelium (thin sides) or the supportive interstitial spaces (thick sides) of the alveolar septa or the visceral pleura, respectively. In both the alveolar and subpleural capillaries, endothelial vesicle numerical densities and the amount of endothelial cell attenuation were greater on the thin sides than on the thick sides. Morphometric comparisons between the subpleural and alveolar capillaries on their respective thin sides defined greater endothelial attenuation (34% of endothelial surface compared to 14%) in the subpleural capillaries. Further, endothelial vesicle numerical densities were less in the subpleural capillaries (118/micron3 compared to 226/micron3). Vesicle diameters, on the other hand, were greater in the subpleural capillary endothelia (0.82 micron compared to 0.64 micron). The differences between subpleural and alveolar capillary endothelia on their respective thick sides were analogous to those described for the thin sides of capillaries. Endothelial attenuation and vesicle diameters on the thick sides of the subpleural capillaries exceeded those on the thick sides of the alveolar capillaries, and subpleural capillary endothelial vesicle densities were less than those of the alveolar capillary endothelia. The marked differences between the alveolar and subpleural capillary endothelia extend previous reports of differences in densities between the capillary networks and serve to further establish regional variations in the pulmonary microcirculation.

Animals↗
PubMed1985

Alveolar microvessels in isolated perfused dog lungs: structural and functional studies after production of moderate and severe hydrodynamic edema.

We have reported earlier that increased endothelial vesiculation follows the development of septal edema and alveolar flooding in isolated dog lung preparations. In this report, established ultrastructural morphometric analyses are coupled with data from physiologic and indicator-dilution studies to evaluate the stage of edema development at which de novo formation of alveolar microvessel plasmalemmal vesicles occurs. The interpretation that alveolar microvessel plasmalemmal vesicles increase prior to alveolar flooding, the final stage of edema formation, is consistent with the results reported here. Moderate pulmonary edema, characterized by substantial fluid cuffing around extra-alveolar arteries and veins and by fluid accumulation restricted to the thick sides of the alveolar septa, is associated with increased vesiculation in alveolar vessel endothelium. Further, a larger percentage of the vesicle population seen is directly attached to the endothelial luminal or abluminal surfaces. The functional significance of an increased population and an altered intracellular distribution of vesicles remains undetermined. The vesicles may provide a minor defense against excessive septal interstitial fluid accumulation, and subsequent alveolar flooding, by contributing to retrograde transport to the blood. Increased vesiculation, on the other hand, may represent an adaptive cellular response to interstitial fluid accumulation.

Animals↗
PubMed1984

Vesicle numerical densities and cellular attenuation: comparisons between endothelium and epithelium of the alveolar septa in normal dog lungs.

The alveolar septa are divided into two anatomically distinct portions: The thin sides consist of capillary endothelium, alveolar epithelium, and their closely apposed (often fused) basal laminae; the thick sides are characterized by prominent interstitial spaces, containing fibrils and cells, which separate the respective basal laminae. Vesicle numerical densities are comparable (approximately 400 vesicles/ micron3 cytoplasm) in the endothelial and epithelial cells on both sides of the septa. Mean vesicle diameters, however, are substantially less in the epithelial cells on both the thin and thick sides. The extent of both endothelial and epithelial attenuation is significantly greater on the thin sides of the septa. Further, epithelial attenuation is more marked than endothelial attenuation on both sides of the septa. The attenuated cellular portions, possibly because of their extreme thinness, are void of vesicles but provide relatively short diffusion distances (20-30 nm) from vessel or alveolar lumen to the basal lamina. Whether these structural differences between endothelial and epithelial cells contribute to physiologic evidence that describes the endothelium as more permeable than the epithelium remains to be established.

Animals↗
PubMed1984

An ultrastructural morphometric comparison of the peripheral with the hilar air-blood barrier of dog lung.

Excised and isolated perfused lungs are frequently used as models for studies of fluid flow and membrane transport. Regional variations of the air and blood vascular compartments of the lung have been recognized to result from gravitational effects. This ultrastructural morphometric study considered the components of the air-blood barriers of the peripheral and hilar regions of excised dog lungs. The lungs of three mongrel dogs were fixed by endotracheal instillation of glutaraldehyde and immersion in fixative. Stratified random sampling, point counting volumetry, and line intercept counts were used to determine the thicknesses of the air-blood barrier and the epithelial, interstitial, and endothelial compartments. Point counting volumetry also established the volume density of the alveolar spaces. The morphometric values for structures within the periphery were statistically compared to those within the hilar region by Student's t-test. Endotracheal and immersion fixation as used in this study, combined with stratified random sampling, equalized regional differences in the alveolar lumen volume and air-blood barrier thickness. With these conditions no significant differences in the volume densities of the epithelial and endothelial cells and the interstitium of the air-blood barrier were identified when those of the hilum were compared to those of the periphery.

Animals↗
PubMed1983

Pulmonary transport of water and solutes: functional and structural correlations.

Assessment of endothelial barrier parameters is based on formulations derived from the thermodynamics of irreversible processes. These formulations contain simplifying assumptions that, with uncertainties concerning the surface area involved in the transport, preclude definitive modeling with respect to the possible pathways. In the development of edema in isolated lung preparations perfused at 37 C, changes of structural features such as increases of vesicle volume density can be associated with changes of barrier parameters such as increased filtration coefficients and increased permeability coefficients to sodium ion. However, that edema can develop at 15 C without increased vesiculation and that cuffing of extraalveolar vessels also occurs in vivo with circulatory overload without increased vesiculation do not suggest that vesicles play a determining role in the development of edema. Available evidence indicates that the exchange of water across the endothelium is at a rate approximating the flow rate of blood and therefore must involve the entire endothelial surface. Separation or noncommunality of pathways for water and solutes makes modeling of the pathways from current formations even less certain.

Animals↗
PubMed1983

Morphometric and physiological studies of alveolar microvessels in dog lungs in vivo after sustained increases in pulmonary microvascular pressures and after sustained decreases in plasma oncotic pressures.

Previous investigations from this laboratory of isolated-perfused dog lungs have shown that volume densities of vesicles in both capillary endothelial and type I epithelial cells are substantially increased after acute, severe edema produced by increasing microvascular pressures (D. O. DeFouw and P. B. Berendsen, 1978, Circ. Res. 43, 72-82) and after decreasing macromolecular concentrations of the perfusate (D. O. DeFouw and P. B. Berendsen, 1979, Microvasc. Res. 17, 90-103). The present study provides morphometric evaluations of alveolar vessel endothelium of the lungs of intact dogs after episodes of sustained increased microvascular pressures and after sustained decreases of plasma oncotic pressures. Endothelial vesicular volume densities did not increase in either case. Fluid accumulation was found in the extraalveolar connective tissue spaces (mainly perivascular); no changes of endothelial or interstitial compartmental thicknesses were observed on either the thin or thick sides of the alveolar septa. Thus, if the increased outward filtration to be expected from the increased hydrostatic or decreased oncotic pressures occurred in the microvasculature, it was moderated by increased hydrostatic and decreased oncotic pressures in the tissues or was accommodated by increased lymph flow rates. Our tentative hypothesis that increased septal interstitial pressure plays a role in initiating vesicle formation in alveolar endothelial cells is consistent with these data if the postulated increases of tissue pressure are less than in the isolated perfused preparations. Our data do not provide support for the concept that endothelial vesicles play a major, early role in the development of pulmonary edema.

Animals↗
PubMed1983

Variations in cellular attenuation and vesicle numerical densities in capillary endothelium and type I epithelium of isolated, perfused dog lungs after acute severe edema formation.

Morphometric comparisons of nonedematous and edematous isolated, perfused dog lungs establish that there are significant differences between the degree of cellular attenuation and vesicle numerical densities in endothelial and type I epithelial cells of the alveolar septa after edema production. In nonedematous isolated lungs the extent of endothelial and epithelial attenuation was greater on the thin sides of the septa. In the edematous lungs, the differential of greater thin-side attenuation was maintained for the endothelium but not for the epithelium where the extent of attenuation in the septal thick segments was increased. Vesicle numerical densities were approximately doubled in the cells on both sides of the septa in the edematous lungs. The endothelial vesicle densities were greater in the septal thin segments than in the septal thick segments in both the nonedematous and the edematous isolated lungs. The epithelial vesicle densities, on the other hand, were similar on the thin and thick sides of the septa in the nonedematous and edematous lung preparations. Although the contribution of vesicles to cellular function in the alveolar septa remains uncertain, further evaluation of vesicular transport should include the possible variability of this function with the varying degrees of cellular attenuation on the two anatomically distinct sides of the septa.

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