Junctional transfer in cultured vascular endothelium: I. Electrical coupling

Vascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (lambda) were 208.6 microns (BAEC) and 288.9 microns (BVEC). The nonjunctional (6.14 and 8.72 X 10(8) omega) and junctional (3.67 and 3.60 X 10(6) omega) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types. In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.     

Junctional transfer in cultured vascular endothelium: II. Dye and nucleotide transfer

Summary Vascular endothelial cultures, derived from large vessels, retain many of the characteristics of theirin vivo counterparts. However, the observed reduction in size and complexity of intercellular gap and tight junctions in these cultured cells (Larson, D.M., and Sheridan, J.D., 1982,J. Cell Biol. 92:183) suggests that important functions, thought to be mediated by these structures, may be alteredin vitro. In our continuing studies on intercellular communication in vessel wall cells, we have quantitated the extent of junctional transfer of small molecular tracers (the fluorescent dye Lucifer Yellow CH and tritiated uridine nucleotides) in confluent cultures of calf aortic (BAEC) and umbilical vein (BVEC) endothelium. Both BAEC and BVEC show extensive (and quantitatively equivalent) dye and nucleotide transfer. As an analogue of intimal endothelium, we have also tested dye transfer in freshly isolated sheets of endothelium. Transfer in BAEC and BVEC sheets was more rapid, extensive and homogeneous than in the cultured cells, implying a reduction in molecular coupling as endothelium adapts to culture conditions. In addition, we have documented heterocellular nucleotide transfer between cultured endothelium and vascular smooth muscle cells, of particular interest considering the prevalence of myo-endothelial junctionsin vivo. These data yield further information on junctional transfer in cultured vascular endothelium and have broad implications for the functional integration of the vessel wall in the physiology and pathophysiology of the vasculature.     

Human monocyte adherence to cultured vascular endothelium: monoclonal antibody-defined mechanisms

We have evaluated the binding of human peripheral blood monocytes to cultured vascular endothelium as an in vitro model of monocyte interaction with the vessel wall. Monocytes were purified (91% +/- 4 SE esterase positive) by elutriation to avoid contact with surfaces before assay. Adherence of 51Cr-labeled monocytes after 45 min (36% +/- 11 SE) was significantly higher than that observed with autologous radiolabeled neutrophils (9% +/- 5 SE) and was greater on monolayers of human umbilical vein endothelium than on bovine aortic endothelium. Peripheral blood mononuclear cells treated with monoclonal antibody (MoAb) 60.3, a reagent that binds leukocyte membrane complex CDw18, implicated in multiple adherence-dependent functions, failed to adhere and flatten on artificial surfaces. Mononuclear cells treated with MoAb 60.3 simulated cells from a patient with recurrent infections whose phagocytes failed to react with MoAb 60.3 and failed to emigrate to extravascular sites in vivo. Incubation of monocytes with MoAb 60.3 inhibited (by 32 to 61%) monocyte adherence to endothelium in a dose-dependent manner for periods up to 24 hr, but had negligible effects on basal (unstimulated) neutrophil adherence. Basal monocyte adherence in the presence of MoAb 60.3 remained significantly greater than basal neutrophil adherence. Augmentation of phagocyte adherence to endothelial monolayers by autologous plasma or phorbol ester (PMA) was abrogated by incubation with MoAb 60.3. Studies with immunofluorescence flow cytometry indicated that PMA stimulation of monocytes resulted in a specific 40% increase in monocyte surface expression of the epitope recognized by MoAb 60.3. These in vitro findings, in conjunction with observations from two patients, support the hypothesis that monocyte adherence to endothelium and emigration to tissues is mediated by mechanisms both dependent upon and independent of the CDw18 complex and the epitope recognized by MoAb 60.3.     

Enhanced adhesion of polymorphonuclear leukocytes to anoxic cultured vascular endothelium

This study showed that the adherence of human polymorphonuclear leukocytes (PMN) to monolayer cultures of human umbilical vein endothelial cells (HUVEC) was increased when the latter were rendered anoxic. This adhesion was greater after 4-5 h than after 8 h of anoxia, but even at 8 h was significantly above the level of adhesion to HUVEC maintained under normoxic conditions for the same period. The changes in adhesion were not dependent on the viability of HUVEC during anoxia. Anoxia-induced adhesion was prevented by addition of cycloheximide (5 micrograms/ml) to the cultures, suggesting that it depended on HUVEC protein synthesis. Enhanced adhesion was also prevented by addition of a monoclonal antibody directed against the cytokine, interleukin-1 alpha (IL-1 alpha). These findings are consistent with a role of endogenous IL-1 alpha as a mediator of the anoxia-induced adhesion of PMN to HUVEC.     

Prediction of LDL concentration at the luminal surface of a vascular endothelium

To find out whether concentration polarization of low-density lipoprotein (LDL) occurs at the surface of a vascular endothelium or not, transport of LDL in flowing blood to an water-permeable endothelium was studied theoretically by means of CFD. Calculations were carried out for an endothelium exposed to a Couette flow by assuming that the surface geometry of the endothelium could be expressed by a cosine function. Two typical cases were considered for the permeability of endothelium to water; one was uniform permeability everywhere in the endothelium, and the other was uneven permeability which was augmented at the intercellular junction. It was found that, in both cases, the surface concentration of LDL increased in going distally from the entrance, taking locally high and low values at the valleys and hills of the endothelium, respectively, and the variation was larger in the case of endothelium with uneven permeability. These results clearly showed that concentration polarization of LDL which might affect the uptake of LDL by the arterial wall certainly occurs at the surface of the endothelium even if the flow is disturbed microscopically by the uneven surface of the endothelium.     

Endocytic vesicles in liver carry polymeric IgA from serum to bile

The distributions both of endogenous IgA and of injected 125I-labelled IgA were determined amongst the components of a liver homogenate. Rate zonal sedimentation, under conditions where separation was principally determined by particle size, showed that IgA was tightly bound to material which sedimented in the size range of the larger endoplasmic reticulum fragments. Further fractionation of the components within this size range according to their densities, by isopycnic centrifugation, showed that the IgA was associated with small vesicles with a density range of 1.12--1.17 g/ml, quite distinct from endoplasmic reticulum fragments. We therefore conclude that the IgA is present in liver cells in a distinct class of vesicles, which are, presumably, responsible for the transport of IgA from blood to bile.     

Organ culture: a new model for vascular endothelium dysfunction

Background  

Endothelium dysfunction is believed to play a role in the development of cardiovascular disease. The aim of the present study was to evaluate the suitability of organ culture as a model for endothelium dysfunction.     

Perivascular nerves and vascular endothelium: recent advances

Within the last few years, advances have been made regarding perivascular nerves and the endothelium of the vascular system, both potentially important in the understanding of the mechanisms of local control of blood flow. Endothelin-1 (ET-1) has been identified in rat cerebrovascular nerves, neuropeptide Y (NPY) has been demonstrated in umbilical endothelium, the arginine-vasopressin (VP) system has been discovered in the heart (including coronary endothelium), and P2X receptors have been observed in vascular endothelial cells. After a brief introduction to vascular biology, this review will focus on the above-mentioned new data.     

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

Ultrafast Ca wave in cultured vascular smooth muscle cells aligned on a micropatterned surface

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which propagates as Ca²⁺ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca²⁺ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca²⁺ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca²⁺]_i dynamics and membrane potential events along the network of cells. The rate of [Ca²⁺]_i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca²⁺ channels (VOCCs) inhibition suppressed the ultrafast Ca²⁺ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca²⁺ entry from the external medium through VOCCs, modeled as an ultrafast Ca²⁺ wave. This wave may trigger and drive slower Ca²⁺ waves observed ex vivo and in vivo.     

A comparative study of thromboxane and prostacyclin release from ex vivo and cultured bovine vascular endothelium

Thromboxane B2, 6-keto-Prostaglandin F1 alpha, and Prostaglandin E2 release have been quantitated from cultured adult bovine endothelial cell monolayers and from ex Vivo vascular segments employing specific radioimmunoassays and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF1 alpha released was diminished compared to amounts released from the vessel segments while thromboxane B2 and prostaglandin E2 release were similar in the two endothelial model systems. However, the amount of thromboxane B2 assayed was small and the quantity of thromboxane A2 it represents is probably of little in vivo significance compared to prostacyclin.     

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium

Cultured bovine aortic endothelial cells were fixed and stained with NBD-phallicidin and quantitated with a digital image analyzer for changes in actin cables and surface area. Serotonin (5-HT), norepinephrine (NE), dopamine and histamine (all at 10(-4)M concentrations) were tested for their ability to induce cytoskeletal changes. Only 5-HT and NE increased actin cables significantly (p less than 0.01), 80.7% and 97.9%, respectively. Dopamine and histamine treated cells showed a 67.4% and 80.8% decrease in actin cables respectively (p less than 0.01). Stimulated increases of actin cables by 5-HT were inhibited by Ketanserin, and propranolol inhibited NE stimulation of actin cables. Treatment of cells with these blockers alone also decreased actin cables below control values (p less than 0.01). Pretreatment of cells with diphenhydramine, but not cimetidine, inhibited histamine-induced decreases in actin cables. Stimulation of surface area by 5-HT and NE was also observed, with 40.8% and 80.7% increases respectively, when compared with controls (p less than 0.01). The increases in actin cables were associated with a lack of ruffled edges that are indicative of motile cells. In contrast, induced decreases in actin cables resulted in cells with ruffled edges. Exogenous 5-HT and NE have been shown to prevent the increased permeability visible as extravasation of red blood cells from postcapillary venules in thrombocytopenic animals. The present data suggest that 5-HT and NE may be involved in maintaining the endothelial barrier function by a receptor-mediated stimulation of actin cables. Also, histamine-induced decreases in actin cables may be correlated with the amine's action in vivo as a mediator of increased inflammatory permeability.     

Differentiation of vascular endothelium in the chick chorioallantois: a structural and autoradiographic study

The maturation of vascular endothelial cells in the chick chorioallantoic membrane, from 8 to 18 days after fertilization, was investigated by light and electron microscopy. Light microscopic autoradiography following administration of tritiated thymidine was used to determine the thymidine labeling index of the endothelial cell population at various stages of development. Results indicate that, prior to day 11 of incubation, endothelial cells have the morphological characteristics of immature and relatively undifferentiated cells. During this time they exhibit a high labeling index of approximately 23%. At 11 days, the labeling index decreases to 2.8%, and subsequently the cells begin to acquire structural characteristics of mature differentiated endothelium. The pattern of endothelial cell labeling suggests that, during the period of high endothelial cell mitosis, the capillary network of the growing chorioallantoic membrane is expanding by an overall proliferation of endothelial cells in existing capillaries, rather than by formation of new capillary sprouts. The immaturity of endothelial cells in the young chorioallantois, or conversely their high rate of cell division, may influence the ability of this membrane to support grafted tissue prior to day 11.