Richard N. Feinberg

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.

APOPTOSIS IN EARLY OCULAR MORPHOGENESIS IN THE MOUSE

Development of the eye requires complex interactions between tissues, extracellular matrix and growth factors. Most cells of the optic primordia grow and differentiate into discrete ocular structures; however, other cells have death as their developmental fate. The most common mechanisms of cell death are apoptosis and necrosis. We have identified the cell death that occurs during ocular morphogenesis in ZRDCT-N mice as apoptosis. Mouse embryos, ages E8.5-E11.5, were embedded in paraffin, sectioned at 5 microns and stained with hematoxylin or by the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The spatial and temporal distribution of apoptotic cells was mapped at 0.5 day intervals using a computerized image analysis system, and 3-D reconstructions were made at each embryonic age. Our data indicate that apoptosis plays a role in normal ocular morphogenesis and provides the groundwork for studies of abnormal ocular development.

Canine jugular vein endothelial cell monolayers in vitro : vasomediator-activated diffusive albumin pathway

Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an in vitro permeability assay. The calculated diffusive permeability coefficient for FITC-BSA across untreated monolayers was 1.1 +/- 0.4 x 10(-6) cm/s. After 15-min incubations with either histamine or bradykinin, the resistance to albumin flux across the monolayers was reduced significantly. Diffusive albumin permeability coefficients were 3.4 +/- 1.8 x 10(-6) and 4.1 +/- 2.0 x 10(-6) cm/s, respectively. Ultrastructural morphometric analyses of the endothelial cell monolayers served to define uniform dimensions of intercellular clefts and similar plasmalemmal vesicle densities in the untreated and the vasomediator-activated monolayers. These results are consistent with the interpretation that the vasomediator-activated pathway across the venous endothelial monolayers is not dependent on sustained intercellular gap formation or sustained expansion of the plasmalemmal vesicle population for the 15-min observation periods. Whether the increased albumin flux is dependent on transient gap formation or on physical changes within the venous endothelial cell glycocalyx remains to be tested.

Responses of Rat Pleural Mesothelia to Increased Intrathoracic Pressure

Direct measurements of pleural fluid hydrostatic and colloid osmotic pressures after infusion of saline, bovine serum albumin, or silicone into the pleural space were coupled with ultrastructural morphometric analyses to assess the response of pleural mesothelial cells to hydrothorax. Increases of hydrostatic pressure, either independent or in combination with decreases of osmotic pressure, served to increase the number of plasmalemmal vesicles in mesothelial cells of both the visceral and parietal pleurae. These results support the hypothesis that an increase in vesicle numerical density represents a response to elevations of extracellular fluid pressures. Fluid resorption from the pleural space with subsequent accumulation within the visceral pleural interstitium was also associated with the formation of invaginations of the mesothelial basal plasmalemma. That the invaginations were not observed in the absence of interstitial fluid accumulation supports the concept that basal surface invaginations represent distortions of mesothelial cell membranes in response to pressure differentials across the plasmalemma. The results of this study are most consistent with the interpretation that increased numbers of plasmalemmal vesicles and invaginations of the basal plasmalemma represent adaptive conformational mechanisms of pleural mesothelial cells to prevent monolayer disruption by elevated extracellular fluid pressures.

Endothelial heterogeneity in the chick wing bud: A morphometric study

SummaryThe microvascular endothelium of the chick wing bud at stages 22, 27, and 32 was evaluated by ultrastructural morphometry. The rationale for this study is based on the hypothesis that endothelial cells exhibit variation in structure and function during cytodifferentiation. The microvessels had a luminal diameter range such that they were classified as capillaries. The thin continuous endothelium was devoid of a basal lamina. The endothelium had a very small number of plasmalemmal vesicles; vacuoles were however present for all stages and in some cases were abundant. The temporal findings were that endothelial cell thickness increases, plasmalemmal vesicle densities decrease, and the densities of cytoplasmic vacuoles increase. The spatial results were that endothelial cells in proximal regions of the limb have a greater thickness, contain fewer vesicles and have more vacuoles than those in distal regions. In general, these results indicate that endothelial ultrastructural heterogeneity occurs within a 31/2 day timespan of wing bud development. The discussion considers the results with regard to recent reports on endothelial cell heterogeneity.

MACROMOLECULAR PERMEABILITY OF CHICK WING MICROVESSELS : AN INTRAVITAL CONFOCAL STUDY

The development of the vertebrate limb requires the formation of a normal vasculature to nurture the soft and hard tissue phenotypes. The pattern of embryonic limb bud vessels has been extensively studied, but little is known about the permeability characteristics of the developing circulation. In the present study, the microvascular endothelial cell phenotype was examined by in vivo confocal microscopy following the systemic injection of a graded series of fluorescent dextrans (40,000, 70,000, 150,000 molecular weight) into chick embryos at stages 21–23 in order to determine how selective is the endothelial lining of microvessels as a partition between the blood vessels and the interstitium. Videodensitometry, over a gray scale range of 0–255, was used to quantitate the amount of tracer found within the interstitial compartment of the limb. The tracers of larger molecular weight (70,000, 150,000) were confined exclusively to the vascular lumina, whereas that of smaller molecular weight (40,000) was found to cause perivascular brightening due to extravasation into the surrounding interstitium. The reported differences in permeability were not dependent upon the stage of the embryo used in this study, but were due to the size of the tracer. These data indicate that embryonic wing microvessels demonstrate permselectivity to macromolecular efflux across the endothelium. The present results provide a basis for additional studies concerned with the dynamic characteristics of the limb microvasculature and challenge our concepts about the role of diffusible morphogens in vertebrate limb development.