Degranulation of mast cells in the chick chorioallantoic membrane does not increase macromolecular extravasation during normal angiogenesis.

OBJECTIVE: To test the hypothesis that stimulation of mast cell degranulation during normal angiogenesis in the chick chorioallantoic membrane (CAM) serves to acutely activate macromolecular transendonelial pathways. METHODS: Using shell-less cultures of 6-day-old chick embryos, efflux of fluorescein isothiocyanate (FITC)-dextran 150 from CAM microvessels was evaluated, after applications of compound 48/80 or exogenous histamine, by computer-assisted videodensitometric analyses. Real-time confocal imaging enabled optical differentiation of first-order pre- and postcapillaries from the capillary networks. Cytologic ultrastructure of the microvascular units was also evaluated. RESULTS: Although endogenous histamine was measurable and its concentration was increased after application of compound 48/80, segmental endothelia of the CAM microvascular units consistently restricted extravasation of FITC-dextran 150. Furthermore, intravascular injections of histamine also failed to induce interendothelial gap formation or macromolecular flux across the segmental CAM endothelia. CONCLUSIONS: These results are consistent with the interpretation that histamine-activated transendothelial pathways are inactive in the CAM at day 6. Whether such inactivity serves to retard facilitation of CAM angiogenesis by activated mast cells 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.

The 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 3 1/2 day time-span of wing bud development. The discussion considers the results with regard to recent reports on endothelial cell heterogeneity.

Vascular histodifferentiation in the chick chorioallantoic membrane: a morphometric study.

This study presents a quantitative description of ultrastructural changes associated with focal histodifferentiation of the chick chorioallantoic membrane (CAM) microvasculature. Early vessels (4-8 days of a 21-day incubation period) were lined by a thin, but continuous endothelium which was characterized by punctate appositional contacts, an incomplete or absent basal lamina, and few plasmalemmal vesicles. During this stage, there was little ultrastructural distinction between CAM arterioles, capillaries, and venules. During the intermediate stage (9-13 days), the arteriolar endothelium displayed more extensive junctional apposition. In addition, the interstitial (periendothelial) space contained more collagen fibrils and increased cell volume densities, some of which resembled presumptive smooth muscle cells. In contrast to the arterioles, endothelial appositions of the venules and capillaries remained punctate during the intermediate stage. The interstitial space of the venules displayed an increase in cell and fibril densities but to a lesser extent than that observed in the arterioles. The capillaries did not exhibit any significant changes in their periendothelial spaces. By the late stage (14-18 days), a circular tunic containing several layers of presumptive smooth muscle cells surrounded only the arteriolar endothelium. The endothelium of both the arterioles and venules increased in mean thickness. The arterioles also displayed complex interdigitating endothelial appositions. All CAM vessels exhibited a progressive increase in the number of endothelial plasmalemmal vesicles and a more complete basal lamina during the developmental stages. These observations served to distinguish differential patterns of vessel maturation along the length of the microvascular system.

Quantitative histochemical evaluation of skeletal muscle ischemia and reperfusion injury.

Acute arterial obstruction to the extremities is associated with significant morbidity and mortality. The evaluation of accompanying skeletal muscle injury has thus far been indirect and imprecise. Triphenyltetrazolium chloride (TTC) is an oxidation-reduction indicator which allows for the histochemical quantitation of skeletal muscle injury. In 21 anesthetized nonheparinized adult mongrel dogs, the isolated in vivo gracilis muscle underwent 4, 6, or 8 hr of ischemia with and without reperfusion. The muscles were excised and cut into 1-cm segments, representative muscle biopsies for electron microscopy were taken, each segment was stained in 1% TTC, and the total area of staining was measured with computerized planimetry. All control muscles stained completely with a dark red color. After 4, 6, or 8 hr of ischemia, quantitative measurements of muscle staining indicative of normal tissue were present in 98 +/- 1%, 59 +/- 5%, and 23 +/- 9% of the total muscle areas, respectively. Six hours of ischemia followed by reperfusion was associated with only 36 +/- 9% of the muscle being stained. Segmental TTC staining demonstrated that reperfusion was associated with greater injury, and less TTC staining, in the proximal portion of the gracilis muscle at the site of entry of the major arterial pedicle. The distal muscle did not demonstrate increased damage with reperfusion. It is hypothesized that protection of the distal muscle from reperfusion injury may be due to an absence of reflow farther away from the artery.

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.

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.