Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

Confluent monolayers of microvascular endothelial cells, derived from the rat epididymal fat pad and grown in culture, were radioiodinated by using the lactoperoxidase method. Their radioiodinated surface polypeptides were detected by NaDodSO4/PAGE (followed by autoradiography) and were characterized by both lectin affinity chromatography and protease digestion to identify the proteins involved in albumin binding. All detected polypeptides were sensitive to Pronase digestion, whereas several polypeptides were resistant to trypsin. Pronase treatment of the cell monolayer significantly reduced the specific binding of radioiodinated rat serum albumin, but trypsin digestion did not. Limax Jiavus, Ricinus communis, and Triticum vulgaris agglutinins competed significantly with radioiodinated rat serum albumin binding, whereas other lectins did not. A single 60-kDa glycoprotein was precipitated in common by these three lectins and was trypsin-resistant and Pronase-sensitive. Rat serum albumin affinity chromatography columns weakly but specifically bound a 60-kDa polypeptide from cell lysates derived from radioiodinated cell monolayers. These findings indicate that the 60-kDa glycoprotein is directly involved in a specific interaction of albumin with the cultured microvascular endothelial cells used in these experiments.The attenuated layer of endothelial cells lining the blood vessels forms the critical barrier controlling the exchange of molecules between the blood and the interstitial fluid. The ability of the endothelium to act both as a passive, albuminmodified barrier to transvascular exchange (1, 2) and as a specific receptor-mediated translocator of molecules [i.e., insulin (3), transferrin (4, 5), and low density lipoprotein (6)] is dependent on interactions occurring at its luminal surface.Through its interaction with the endothelium, albumin has several important vascular functions. As the major blood protein, it is the principal determinant of the oncotic pressure of the plasma and thereby strongly influences transendothelial fluxes of water and small solutes. In addition, its interaction with the endothelial glycocalyx (2,7,8) apparently creates a permselective barrier that limits the transendothelial passage of many molecules (1, 9). Upon removal of albumin from vascular perfusates, for instance, apparent solute and water permeabilities increase significantly while osmotic reflection coefficients decrease in both individually perfused capillaries (1) and whole organ preparations (2, 10).

Capillary endothelial cells have a large population of small (65-80 nm diameter in transmission electron microscopy) vesicles of which a large fraction is associated with the plasmalemma of the luminal and abluminal side. We studied the fine structure and distribution of these plasmalemmal vesicles by high resolution scanning electron microscopy in cultured endothelial cells obtained from bovine adrenal cortical capillaries. Cell monolayers were covered with polylysine-coated silicon chips, split in high potassium buffer, fixed in aldehyde mixtures, and then treated with OsO4 and thiocarbohydrazide. After critical point drying, the specimens were coated with a thin (<2 nm) continuous film of chromium. On the cytoplasmic aspect of the dorsal plasmalemmal fragments seen in such specimens, plasmalemmal vesicles appear as uniform vesicular protrusions ~70-90 nm in diameter, preferentially concentrated in distinct large fields in which they occur primarily as single units. Individual plasmalemmal vesicles exhibit a striped surface fine structure which consists of ridges ~10 nm in diameter, separated by furrows and oriented as meridians, often ending at two poles on opposite sides of the vesicles in a plane parallel to the plasmalemma. This striped surface structure is clearly distinct from the cage structure of coated pits found, at low surface density, on the same specimens. The cytoplasmic aspect of the plasmalemma proper is covered by a fibrillar infrastructure which does not extend over plasmalemmal vesicles but on which the latter appear to be anchored by fine filaments.The permeability of blood capillaries provided with a continuous endothelium has been explained by convection and diffusion along intercellular junctions (1, 2) and transendothelial channels (3) and by transcytosis, i.e., vesicular transport across the endothelium via plasmalemmal vesicles (4-7). Available morphological and functional evidence supports transcytosis as an exchange mechanism across the endothelium of such capillaries (3, 8), but the surface and volume density of plasmalemmal vesicles (9), their relation to the plasmalemma proper and to one another, and especially their presence as free units within the endothelial cytoplasm are still debated (10-12; see, however, 13).To gain additional information on plasmalemmal vesicles, we have adsorbed the plasmalemma of cultured endothelial cells to a solid substrate, split the cells to expose the cytoplasmic aspect of the adsorbed membrane, and examined it by high resolution scanning electron microscopy SEM ' (14). The results indicate that plasmalemmal vesicles have a characteristic surface structure distinct from that of coated pits and plasmalemma proper. They also show that in the specimens examined these vesicles occur primarily as single units rather than chains or dendritic structures. We assume that these findings will be of use in further studies on the physiological role of plasmalemmal vesicles and on the mechanisms involved in transcytosis.Part of this work was presented ...

CBX is able to inhibit rapidly the generation of active GC in human adipose tissue. Importantly, limiting GC availability in vivo has functional consequences including decreased glycerol release.

The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degrees C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at approximately 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the endothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.