Three-dimensional analysis of vacuoles and surface invaginations of capillary endothelia in the eel rete mirabile.

One layer of attenuated endothelia of continuous capillaries provides a partially selective diffusion barrier between the blood and the interstitium. Ultrastructures of membrane specialization without the known physiologic functions have been found in blood vessel endothelia. The vacuolar profiles or vacuole-like, membrane-bound structures, which are larger than plasmalemmal vesicles, have been observed routinely in normal endothelial cytoplasm or in blood vessels challenged by insults in electron microscopic studies. Three-dimensional information from serial sections is required to understand the organization and functions of vacuole-like structures in capillary endothelium. The capillaries in eel retia mirabile were perfused with electron-dense tracers, glutaraldehyde in buffer, and were processed for transmission electron microscopy. Ribbons of serial thin sections without counterstaining were examined under a transmission electron microscope. The vacuolar profiles inside endothelial cytoplasm were investigated with the techniques of serial section analysis and three-dimensional reconstruction from serial sections. The vacuole-like structures inside endothelial cytoplasm either were connected to extracellular (luminal, abluminal) compartments or existed as isolated vacuoles from serial section analysis. In the eight series examined in this study, six of ten vacuole-like structures were classified as isolated vacuoles inside endothelia, and their diameters ranged between 186 nm and 266 nm. Two of ten vacuole-like structures were found to extend to the luminal surface of capillaries as luminal, pocket-like invaginations. One of ten vacuole-like structures was found to be connected to the albuminal compartment, and another one existed as an extracellular compartment surrounded by endothelia. Three-dimensional projection of the vacuolar compartments from serial sections showed that endothelial cytoplasm of sheet shape protruded and folded over adjacent endothelium. Three-dimensional information from serial sections reveals the organization of vacuolar profiles and pocket-like invaginations from the cell surfaces in capillary endothelium. The vacuolar profiles in capillary endothelia in two-dimensional electron photomicrographs may represent the extracellular compartments surrounded by the endothelial finger-like extensions. The results indicate that the luminal and abluminal surfaces of the capillary lumen are not smooth or static, and endothelia may change their shape in three dimensions through cytoplasmic protrusions when the tissue environment changes.

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. METHODS: Pericytes cultured from isolated rate capillaries were subjected to angiotensin II and histamine. Their response was monitored by measuring the area of non-yielding substrate covered by the pericytes and the manner in which their shape changed. Shape changes were quantified by calculating the surface area: perimeter perimeter ratios. RESULTS: Histamine significantly reduced surface area covered and the surface area:perimeter ratio. The pericyte processes retracted, resulting in elongated, spindle-shaped cells. These effects were nullified by the H1 blocker diphenhydramine suggesting a receptor-specific response. Angiotensin II also elicited contraction and reduced surface area, but the cells contracted laterally and longitudinally. The surface area: perimeter ratios also decreased. CONCLUSIONS: These results indicate that pericytes are capable of two types of contractile responses in culture, depending on the specific vasoactive stimulus.

SEM of capillary pericytes prepared by ultrasonic microdissection: evidence for the existence of a pericapillary syncytium.

Retia mirabile of the eel swimbladder were exsanguinated, perfusion-fixed and subjected to prolonged osmication. They were then microdissected by ultrasonication which delaminated the capillary bed along planes which revealed the surfaces of arterial and venous capillaries. This procedure resulted in cleaned capillary surfaces largely free of connective tissue elements and basement membrane material. The arterial capillary segments were heavily invested with pericytes characterized by plump cell bodies containing nuclei and an extensive system of processes encircling the capillary wall. These processes exhibited a hierarchical organization consisting of primary, secondary, and tertiary elements arising roughly at right angles to each other. Primary and secondary processes exhibited frequent anastomoses and resulted in cytoplasmic continuity between adjacent cell bodies. Processes were also observed to form connections between pericytes on adjacent capillaries. These observations are evidence for the existence of a pericapillary syncytium in which cell bodies may be connected in series and in parallel throughout the arterial capillary bed. This syncytial organization would provide for a coordinated and global contractile response of pericytes to vasoactive hormones and other effectors. It may also provide for synchrony of nuclear division during developmental spread of pericytes along capillary surfaces.

Cryofixation of vascular endothelium.

Cryofixation refers to the immobilization of tissue components by the rapid removal of heat from the specimen, so that the structure is interred and stabilized in a natural embedding medium, namely, frozen (amorphous or microcrystalline) tissue water. Cryofixation is now often used as a complement to the more traditional fixation methods, especially when the cell structure is delicate or dynamic and may be inaccurately preserved by the slow selective action of chemical fixatives. Vascular endothelial cells are specialized for transcellular transport and for the regulation of blood flow and composition. The dynamic and labile subcellular organization of these cells, presumably reflecting these functional specializations, makes them ideal candidates for cryofixation. Several different types of endothelial cells were directly frozen at temperatures below 20 degrees Kelvin by pressing them against a liquid-helium-cooled block. These samples were subsequently processed for structural analysis by freeze-substitution. Detailed rationales, designs, and protocols are described for both freezing and freeze-substitution. Electron micrographs of cryofixed arterial and venous capillaries (rete mirabile of the American eel), iliac vein (rabbit), and cultured endothelium from the iliac vein (human) reveal that the organization of the characteristic intracellular membrane system of endothelial vesicles is qualitatively similar to that seen in chemically fixed endothelium, especially with regard to the interconnection of clusters of individual vesicles to form elaborate networks. The luminal and abluminal networks are not in communication, at least not in static images. Quantitatively, however, most directly frozen endothelial cells have far fewer vesicular profiles than comparable glutaraldehyde-fixed cells. The differences can be explained by presuming that the rapid action of cryofixation (approximately 1 msec) gives a more accurate picture of the vesicular network because it captures the transient structure of labile or dynamic membranes.

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.

Morphometric changes in pericyte-capillary endothelial cell associations correlated with vasoactive stimulus.

The arterially-derived capillaries of the eel rete mirabile are heavily invested with highly arborized pericytes. By perfusing vasoactive agents through these capillaries, measuring changes in outflow volume and analyzing alterations in capillary and pericyte morphology a set of vasoactive agent-correlated changes were quantified. Morphometric analysis of the capillary ultrastructure revealed that alterations in flow and appearance were associated with changes in the extension of pericyte processes. These responses of the arterial pericytes to the vasoactive agents used in this study suggest that pericyte contraction alters the architecture of the arterially-derived capillaries of the rete in a manner which affects their permeability.

Ultrastructural distribution of terbium across capillary endothelium: detection by electron spectroscopic imaging and electron energy loss spectroscopy.

We used terbium as an intravital tracer of permeability pathways across the walls of capillaries in the rete mirabile of the eel swimbladder and in frog mesentery. Terbium was detected in unstained ultra-thin sections by electron density using electron spectroscopic imaging (ESI) and by electron energy loss spectroscopy (EELS). Enhancement of intrinsic contrast in zero loss images (elastically scattered electrons) permitted imaging of membrane-bound compartments and terbium within them which might otherwise have been undetected in counterstained sections. Element-selective imaging with EELS indicated that terbium was associated with heavy electron-dense deposits, but the terbium mass:volume of sections in areas of lighter deposition was insufficient to obtain a terbium signal. In the rete capillaries, terbium was deposited on the luminal surface, throughout vesicular profiles, and in the interstitium, but could not be traced through interendothelial junctions. Fine terbium deposits were detectable throughout apparent vesicular connections across the endothelium. In the frog mesentery, terbium penetrated some but not all interendothelial clefts, and was detectable in small quantities within luminal and abluminal vesicular profiles and in the interstitium. The results indicate that in the rete capillaries, terbium permeates the capillary via a transcellular route. This route may be provided by transient fusions of luminal and abluminal vesicular compartments.

Associations between pericytes and capillary endothelium in the eel rete mirabile.

Morphometric analysis of electron micrographs of the eel rete mirabile revealed that pericytes occupied nearly one-third of the cellular volume of this organ with over 75% of the pericyte volume associated with arterially derived capillaries. These pericytes were highly arborized, extending processes which encircled the capillaries and covering, on average, more than 85% the ablumenal surface of arterially derived capillaries. Pericytes and endothelial cells interacted in a manner suggesting that pericyte activity modulates both capillary blood flow and permeability.

Preparation of isolated blood capillaries.

Blood capillaries have been isolated from various tissue sources yielding suspensions of capillary segments. These have provided opportunities to study the cellular properties of capillary endothelium under conditions uncomplicated by the presence of stromal tissues and in which measured parameters can be attributed to endothelial cells. Fresh capillary isolates have been used directly as experimental systems but the yield of endothelium is quite low. Amplification of endothelial biomass has been accomplished by using freshly isolated capillaries as explants for primary tissue culture. It has not been previously possible, however, to obtain large amounts of capillary endothelium from a single preparation nor have different capillary types been isolated from the same tissue. The rete mirabile of the eel swim bladder is a copious source of capillaries of two types: thick-walled, continuous capillaries heavily invested with pericytes and thin-walled, fenestrated capillaries. These can be isolated in large numbers free of large blood vessels and contaminating stromal tissue. The two types of capillaries can be isolated from each other by perfusing magnetic beads into one type prior to isolation and separating them from the other type in a magnetic field. This provides a system in which the cellular properties of the two types of endothelium can be studied in vitro and, due to a common isolation procedure, direct comparisons can be made.

Three-dimensional organization of the plasmalemmal vesicular system in directly frozen capillaries of the rete mirabile in the swim bladder of the eel.

Several recent studies comparing chemically fixed and cryofixed endothelium have indicated that glutaraldehyde fixation may result in increases in the population of "vesicles" in the cytoplasm. Other reports based on ultrathin serial-section reconstruction of chemically fixed endothelium have revealed that the vesicular system is comprised of interconnected membranous compartments, which are ultimately continuous with either cell surface but do not extend across the endothelial cell. In this study, we have investigated the three-dimensional organization of the vesicular system in directly frozen, freeze-substituted capillaries of the rete mirabile from the swim bladder of the eel, specifically using the same block of embedded capillaries in which frozen capillaries had previously been found to contain less "vesicles" than chemically fixed capillaries. The results show that essentially all vesicles remain interconnected with each other and are part of two separate sets of invaginations from the luminal and abluminal cell surface like in chemically fixed tissue. Any increase in vesicle number resulting from glutaraldehyde fixation does not affect the overall three-dimensional organization of the vesicular system in these endothelial cells.

Ultrastructure of capillaries in the red body (rete mirabile) of the eel swim bladder.

The retea mirabilia are paired capillary organs located on the dorsal surface of the swimbladder of the common eel. They consist of bundles of closely apposed capillary segments which function in countercurrent exchange of gases and other solutes and concentrate oxygen in the swim bladder. Ultrastructural features of the afferent arterial capillaries and efferent venous capillaries were studied by scanning EM of corrosion casts and critical-point dried retes and transmission EM of thin sections and freeze-fractured retes. A loose association between endothelial cells in the venous capillaries is indicated by penetration of casting material into interendothelial clefts and the appearance of clefts bounded by cytoplasmic flaps in exposed critical-point dried specimens. In thin sections, open gaps between venous endothelial cells are bounded by cytoplasmic processes. Sections through arterial capillaries exhibit tight occluding junctions joining endothelial cells together and these can be seen in freeze-fracture replicas to extend without interruption along the length of the arterial capillaries. These studies indicate the absence of open or hydraulically conductive pathways across the arterial capillary walls and that they probably constitute a rate-limiting barrier in countercurrent exchange of solutes.

Mechanism of sucrose uptake by isolated rat hepatocytes.

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C-sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration-dependent from 5 microM to 100 mM and follows first-order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid-phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.

Rapidly-frozen, cultured, human endothelial cells: an ultrastructural and morphometric comparison between freshly-frozen and glutaraldehyde prefixed cells.

Cultured, human endothelial cells from the iliac vein were divided into two groups for rapid-freezing using liquid helium in a Heuser-type cryopress freezing machine. The first group of cells was prefixed in buffered 2% glutaraldehyde solution whereas the second group was freshly-frozen. Following freeze substitution and embedding, morphometric analysis was carried out on thin sections of groups of endothelial cells. Measurements showed that glutaraldehyde prefixation significantly increases the numerical and volume densities of the vesicles compared to those of the freshly-frozen cells. However, the fact that no significant increase was demonstrated either in the numerical or volume densities of the caveolae, and that no significant change was observed in the ratio of the cell perimeter to area, suggests that the membrane source for these extra vesicles is internally generated. Studies are currently under way to identify the source of this membrane.

Differential endocytosis of lipoproteins by capillary endothelial vesicles.

Plasma levels of lipoproteins are believed to be controlled largely by lipoprotein receptors on the surfaces of tissue cells which facilitate their internalization and degradation. This presupposes transit of lipoproteins across the walls of blood vessels in order to gain access to the receptor sites. The endothelium of tissue capillaries may therefore constitute an additional regulatory barrier for lipoproteins. In order to test this hypothesis, we have measured the uptake of fluorescent-labeled lipoproteins into endothelial vesicles of capillaries isolated from fat tissue. HDLs are ingested at more than twice the efficiency of LDLs and VLDLs are excluded from vesicular ingestion. This represents a decreased efficiency of ingestion with an increase in molecular diameter of lipoproteins. This phenomenon correlates well with the dimensions of endothelial vesicles and caveolae which may restrict entry of very large serum lipoproteins. Selective transport of lipoproteins by capillary endothelial vesicles on the basis of molecular size may therefore serve to regulate blood-tissue interchanges of lipid.

Transport of immunoglobulin G by endothelial vesicles in isolated capillaries.

The ability of capillary endothelial vesicles to transport immunoglobulin G (IgG) across the capillary wall was investigated. Isolated capillary endothelial cells were found to endocytose fluorescent-labeled IgG via a bulk fluid-phase mechanism. When placed in marker-free medium the cells released all previously ingested label. Cationization of IgG causes greater amounts of the protein to be ingested as compared to normal IgG. Cationized IgG is probably endocytosed via an adsorptive mechanism, allowing greater efficiency in its uptake. It is concluded that IgG can be transcytosed by isolated endothelial cells of capillary origin and that the charge of the IgG molecule is important in determining its mode of uptake.

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.