Roger C. Wagner

The isolation and culture of capillary endothelium from epididymal fat

Abstract Capillaries were isolated from collagenase-dissociated epididymal fat by centrifugation to remove floating adipocytes and subsequent filtering to remove blood elements and free stromal cells. Isolated capillaries were cultured in Medium 199 and 20% fetal calf serum. Spreading of endothelial cells from the capillary explants could be directly observed. Nonendothelial cells were removed from primary cultures by treatment with thimerosal. A small percentage of the cells spreading from the explants exhibited cusp-shaped or circular profiles, which were no longer observed after the cells had spread into monolayers. Purified primary cultures consisted of sheets of close-packed polygonal cells. Electron microscopy revealed that micropinocytic vesicles were present in abundance both at the cells free surfaces and enclosed within the cytoplasm. Endothelial cells were frequently joined by intracellular junctions. Weibel-Palade bodies were not evident in those cells observed by electron microscopy. Upon subculturing, the cells progressively exhibited pleomorphism.

The effect of tannic acid on electron images of capillary endothelial cell membranes

Treatment of endothelial cells with tannic acid-glutaraldehyde results in an increase in thickness and electron density of the outer leaflet of surface membranes. The dependency of this effect upon subsequent staining with heavy metals indicates that enhanced density results from binding of heavy metals to sites of tannic acid deposition at the cell surface. When cells are treated with tannic acid after fixation in glutaraldehyde and osmium tetroxide, both inner and outer leaflets of surface membranes and intracellular membranes exhibit an increase in thickness and electron density. This effect is likely due to permeation of osmicated membranes by tannic acid and indicates that tannic acid is able to associate with both surfaces of cellular membranes and acts as a mordant, facilitating heavy metal binding which results in enhanced density and contrast. Uranium and lead are more effective in imparting electron density at sites of tannic acid deposition han osmium.

Phase transition characteristics of diphosphatidylglycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers: Mono- and divalent metal ion effects

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.

Transcapillary transport of solute by the endothelial vesicular system: Evidence from thin serial section analysis

Upon crossing the walls of continuous capillaries, perfused electron-dense solutes often appear to become lodged in the attenuated space bounded by pericytes and the ablumenal capillary surface. In single thin sections, it is difficult to determine whether such accumulations result from transcellular transport via the endothelial vesicular system or whether tracer has filled these spaces retrogradely after paracellular transport through interendothelial clefts. We have taken continuous thin serial sections through patches of terbium trapped between pericytes and the endothelial cells of continuous capillaries in the rete mirabile of the eel. Sampling these areas in three dimensions has revealed that the terbium deposits are bounded and limited to regions of the capillary wall devoid of interendothelial associations. Dense interstitial terbium deposits were also continuous with ablumenal endothelial caveolae and could also be seen in clusters of fused vesicles. These observations strongly implicate transcellular transport by the endothelial vesicular system as the route by which accumulated terbium had crossed the capillary wall.

Metabolic studies on the micropinocytic process in endothelial cells

Abstract Tufts of capillary endothelium isolated from rat epididymal fat ingest [ 14 C]sucrose by the process of micropinocytosis. Ingestion of sucrose for periods in excess of 30 min was quantitated using the concentration of cellular DNA as a standard. Capillaries incubated in the metabolic inhibitors 2,4-dinitrophenol, NaN 3 , or iodoacetate exhibit a dose-dependent reduction of cellular ATP. Micropinocytic ingestion of [ 14 C]sucrose is not affected by concentrations of these inhibitors which markedly reduce or deplete ATP pools. Pure oxygen or nitrogen atmospheres also have no effect on micropinocytosis. Ingestion of [ 14 C]sucrose is reduced, however, at temperatures lower than 37° or in the presence of Zn 2+ .

Differential contractile response of cultured microvascular pericytes to vasoactive agents.

Objective: Microvascular pericytes may contract in two different ways: In the first, a circumferential or radial mechanical force applied at right angles to the long axis of the vessel may constrict the underlying vessel affecting blood flow and transmural pressure. Retraction and elongation of pericyte processes may also occur tangentially and at right angles to the vessel axis and alter microvessel permeability by changing the amount of ablumenal surface covered or the openness of interendothelial junctions. In this study, cultured pericytes were utilized as a model experimental system to determine if vasoactive stimulation changes their shape in a manner consistent with this hypothesis.

Ultrastructure of the vesicular system in rapidly frozen capillary endothelium of the rete mirabile

Abstract Capillaries in the rete mirabile of the eel swim bladder were frozen at liquid helium temperature with a metal-mirror, rapid-freezing device. These were compared to similarly frozen preparations which had been previously fixed with glutaraldehyde. The volume density of endothelial vesicles was three times greater in glutaraldehyde-fixed capillaries than in fresh-frozen capillaries. The number of caveolae per unit of surface membrane was also substantially greater in glutaraldehyde-fixed specimens. No accompanying changes in the total surface membrane or cytoplasmic volume was evident which might account for additional vesicles. Freshly frozen capillaries were characterized by a population of minivesicles (20- to 30-nm diameter) and cisternal cytoplasmic membrane systems which were absent or scarce in glutaraldehyde-fixed capillaries. It is hypothesized that fusion of minivesicles and subdivision of cisternal membrane systems in the presence of glutaraldehyde result in a spurious increase in the endothelial vesicle population. Rapid-freezing thus appears superior to glutaraldehyde fixation in preserving the distribution and form of endothelial vesicles in capillaries.

Endocytosis and exocytosis of transferrin by isolated capillary endothelium

Vesicular transport of solutes across capillary walls may be regulated by specific solute-endothelial interactions. Little data is available on the vesicular transport of serum proteins which may transit the capillary wall in situ. Capillaries were isolated from epididymal fat and incubated in fluorescent-labeled transferrin and radiolabeled sucrose. Endocytosis and exocytosis of these tracers were quantitated on a picomolar basis over timed intervals and standardized against the amount of endothelial DNA present in the isolate. The rate of vesicular endocytosis of transferrin was 6-7 times greater than that of sucrose indicating a mechanism of selection for transferrin. Endocytosis as a function of external concentration exhibited complex kinetics for transferrin that was consistent with an adsorptive component and a fluid component. Sucrose uptake appeared to be simple fluid endocytosis but with a rate-limiting concentration at 500-600 microM. Vesicular exocytosis of both solutes from preloaded capillaries appeared to occur more rapidly than their endocytosis. This was probably not due to different rates of filling and emptying of attached vesicles nor to an intrinsic difference in rates of vesicle interiorization and refusing with the plasma membrane. Different rates of endocytosis and exocytosis may only be apparent since exocytosis of marker before the capillaries reach ingestion equilibrium would reduce the measured uptake rate.

Regulation of micropinocytosis in capillary endothelium by multivalent cations.

Abstract Capillary endothelium isolated from rat epididymal fat exhibits the ability to ingest [ 14 C] sucrose by micropinocytosis. Stimulation of micropinocytosis was observed when the external concentration of either Ca 2+ or Mg 2+ was increased to 10 m M . Other divalent and trivalent cations had either no effect or were inhibitory dependent upon their external concentration. These cations, at an external concentration of 10 m M , inhibited micropinocytosis with the following order of effectiveness La 3+ > Sr 2+ ⪰ Tb 3+ > Ba 2+ . Removal of divalent cations from the incubation medium by chemical chelation resulted in the stimulation of micropinocytosis. Microvesicular efflux of previously ingested [ 14 C]sucrose was stimulated by Ca 2+ and inhibited by the presence of EDTA or at reduced temperatures. Micropinocytic activity in isolated endothelium involves both microvesicular endocytosis and exocytosis. These two concurrent processes, necessary for vesicular transport, appear to be regulated by cations in dissimilar ways.

High-voltage electron microscopy of capillary endothelial vesicles ☆

Conventional electron microscopy of thin sections through capillary walls is inadequate to discern the relationships between endothelial vesicles and their association with the cell surface. High-voltage electron microscopy of thick sections through diaphragm muscle capillaries has been employed to visualize the three-dimensional structure of the vesicular system. Stereopairs of thick sections provide for direct three-dimensional observations of samples several vesicle diameters deep. A variety of simple and compound vesicular forms are present but not all are conjoined or maintain connections with the endothelial cell surface. This contributes to the concept of a dynamic interaction between free and attached vesicular structures where fission and fusion events compartmentalize and reconnect repeatedly. Such interactions would provide for a discontinuous pathway across the capillary wall but with a higher degree of complexity than a simple shuttling mechanism.