Molecular sieving characteristics of the cultured endothelial monolayer

We examined the selectivity of the bovine pulmonary artery endothelial monolayer in vitro to molecules of different sizes. The cultured bovine pulmonary endothelial monolayer was grown on a gelatinized filter and the transendothelial transport was studied by determining the permeability of molecules ranging from 182 to 340,000 daltons under diffusion conditions. The permeabilities across the cultured bovine endothelium were modeled according to cylindrical pore theory. The data were best fit by a two-pore model with radii 65 A and 304 A and a ratio of small to large pores of 160:1. The results indicate that the cultured endothelial monolayer is a selective barrier to molecules of different sizes and that the molecular selectivity is consistent with a diffusional pathway through endothelial pore equivalents. The cultured endothelial monolayer is a useful system for studying the permeability characteristics of the endothelial barrier.     

Transendothelial transfer of macromolecules in vitro

The transendothelial transfer of macromolecules has been difficult to study because of the complexities of the in vivo models. We have developed a model of an endothelium cultured on a permeable support and used it to characterize the transendothelial transfer of albumin. Porcine pulmonary artery endothelial cells form a single layer of cells lining the gelatin-impregnated polycarbonate micropore filters, and the cells develop junctional structures similar to endothelial tight junctions observed in vivo. The monolayer resists the flow of electrical current, and the resistance is sensitive to extracellular calcium concentrations. Albumin transfer across the cultured monolayers was found to be asymmetric, and the rate of transfer from interstitium to lumen was greater than that from lumen to interstitium. The asymmetric transfer occurred against a concentration gradient and was abolished by treating the monolayer with NaCN. Increasing albumin concentrations increased the rate of interstitial to luminal transfer, and the process demonstrated saturation at an interstitial albumin concentration of 725 microM. These data point out the usefulness of the in vitro preparation to identify potentially important aspects of transendothelial transport that would be difficult to detect in vivo.     

Endothelial monolayer permeability to macromolecules

The barrier function of the endothelial monolayer has not been extensively investigated using the cultured endothelium. The in vitro approach may contribute to a more complete understanding of microvessel wall permeability. Our studies using an in vitro endothelial monolayer system have led us to the following conclusions: the endothelial monolayer is more permeable to small-molecular-weight substances than to large molecules; the permeability of albumin is different for endothelial cells derived from different vascular sites (higher for pulmonary venous than pulmonary arterial endothelium); basement membrane components may have a significant role in the permeability of albumin across the endothelium; control of endothelial monolayer permeability is determined not only by the characteristics of the macromolecule (i.e., size and charge) but also by the shape of the endothelial cells and the size of interendothelial space.     

Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current

Bovine microvascular endothelial cells (BMECs) proliferated to confluence on the stromal surface of human amniotic membrane that had been denuded of its natural epithelium. The resulting cultures had the following characteristics: (a) The endothelial cells formed a thin, continuous monolayer and, like their in vivo counterparts, contained basal adhesion plaques and large numbers of cytoplasmic vesicles and 10- nm filaments. In addition, the endothelial cells elaborated a basement membrane-like structure. (b) The borders of the BMECs reacted with AgNO3 to produce the "flagstone" pattern typical of endothelium stained with this reagent in vivo. (c) More than 90% of the zones of contact between endothelial cells examined 8 d after plating prevented passage of a macromolecular probe (wheat germ agglutinin conjugated to horseradish peroxidase) across the BMEC monolayer. (d) 8 d-old cultures displayed a transendothelial electrical resistance that averaged 69 +/- 28 omega X cm2. Monolayers of BMECs maintained on amnion thus resemble in vivo endothelium in several respects and should provide a useful and relevant model for the in vitro study of various phenomena that occur at the microvascular wall.     

An in vitro system for measuring endothelial permeability under hydrostatic pressure

A system was developed, using early passage porcine aortic endothelial cells cultured on a microporous substratum mounted in a two-compartment chamber. It allows the application of a transendothelial pressure gradient and quantitative measurement of the resulting flow rate of fluid. Initial application of a hydrostatic pressure gradient of 20 mmHg resulted in a continuous decrease in the flow rate which reached a steady state after a period of 1-3 h. Further variations in the pressure resulted in pressure-dependent increase or decrease in the flow rate. The physiological relevance of this response is supported by the fact that decrease in permeability occurred only in the presence of Ca2+ ions. Removal of Ca2+ from a monolayer by EGTA led to an immediate increase in the flow rate, whereas readdition of Ca2+ in concentrations between 0.5 and 20 mM was observed to cause a concentration-dependent decrease in flow rate. Initial application of pressure with Ca2+-free medium failed to produce permeability changes of the cultured endothelium. These findings indicate that the permeability of a cultured endothelium to water and solutes is pressure- and Ca2+-dependent.     

Increase in permeability of human endothelial cell monolayer by recombinant human lymphotoxin

The effect of lymphotoxin (LT) on transendothelial permeability was examined using in vitro human endothelial cell (EC) culture system. To assess permeability, we measured the movement of human IgG labeled with horseradish peroxidase (HRP-huIgG) across an EC monolayer cultured on a fibronectin and collagen-coated membrane. LT increases the permeability dose dependently within 16 hours. Similar results were obtained with tumor necrosis factor (TNF)-alpha. The results suggest that increase in vascular permeability by LT and TNF-alpha plays an important role in initiating hemorrhagic necrosis of tumors in vivo.     

Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity

Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.     

Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.     

Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has been shown to increase potently the permeability of endothelium and is highly expressed in breast cancer cells. In this study, we investigated the role of VEGF/VPF in breast cancer metastasis to the brain. Very little is known about the role of endothelial integrity in the extravasation of breast cancer cells to the brain. We hypothesized that VEGF/VPF, having potent vascular permeability activity, may support tumor cell penetration across blood vessels by inducing vascular leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels. We observed that VEGF/VPF significantly increased the penetration of the highly metastatic MDA-MB-231 breast cancer cells across the HBMEC monolayer. We found that the increased transendothelial migration (TM) of MDA-MB-231 cells resulted from the increased adhesion of tumor cells onto the HBMEC monolayer. These effects (TM and adhesion of tumor cells) were inhibited by the pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor (KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can modulate the TM of tumor cells by regulating the integrity of the HBMEC monolayer. Taken together, these findings indicate that VEGF/VPF might contribute to breast cancer metastasis by enhancing the TM of tumor cells through the down-regulation of endothelial integrity.     

Effects of alterations in endothelial cell volume on transendothelial albumin permeability

We examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C-urea) decreased twofold and remained decreased over a 30-minute time-span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125I-albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125I-albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.     

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.     

Effects of calcium on transendothelial albumin transfer and electrical resistance

Nicolaysen, and more recently Kern and Malik, reported that chelation of calcium increased microvascular hydraulic conductivity and albumin permeability in isolated perfused lungs. To begin to understand how calcium affects endothelial function we examined the effect of calcium chelation on an in vitro endothelium. Chelation of calcium with ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid increased the rate of transendothelial albumin transfer by 125%. Reincubation of the endothelium in calcium-repleted medium restored the rate of transfer to its original value. Chelation of extracellular calcium abolished transendothelial electrical resistance. The transendothelial electrical resistance was also restored to normal by reincubation of the endothelium in calcium-repleted medium. Chelation of extracellular calcium caused adjacent endothelial cells to retract from one another, and normal apposition of adjacent cells was restored after reincubation in calcium-repleted medium. Chelation of extracellular calcium produced a centripetal retraction of the peripheral band of actin in individual endothelial cells, and the actin band resumed its normal location after reincubation in calcium-repleted medium. Calcium is an important determinant of endothelial integrity and alterations in calcium produce dynamic changes in endothelial barrier properties and in endothelial-cell shape.     

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm².h and 0.385 μg/cm².h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.     

Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of na?ve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased na?ve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.     

Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34(+) cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34(+) cells in response to stromal cell-derived factor-1alpha. Stromal cell-derived factor-1alpha-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34(+) cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.     

Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells

Rates of fluid pinocytosis by bovine aortic endothelial cells were measured during various manipulations of growth status in vitro. Sparsely seeded cultures grew exponentially until a confluent monolayer was formed, at which time growth slowed. This change in growth rate coincided with a decline in the rate of pinocytosis to about one-third that in the growing cultures. During the subsequent attainment of maximal cell density in the confluent monolayer, the pinocytic rate remained constant. There was close correlation between ³H-thymidine labelling indices, as measured by autoradiography, and the rates of pinocytosis. Mechanical “wounding” of the confluent monolayer resulted in cell migration and proliferation. Twenty-four hours after “wounding,” rates of pinocytosis per mg. cell protein were significantly enhanced. When regeneration of the monolayer was blocked by cytochalasin B, pinocytosis remained at the same rate as in the uninjured, confluent monolayer. These experiments support, and extend to endothelium, earlier observations that in growing cells pinocytosis proceeds at a higher rate than in non-growing, quiescent cells. Furthermore, they raise the possibility that the transendothelial transport of macromolecules such as lipoproteins by receptor-in-dependent fluid pinocytosis in vivo may be altered by the growth status of the endothelium.     

Reorganization of endothelial cells cytoskeleton during formation of functional monolayer in vitro

Endothelium lining the inner surface of vessels regulates permeability of vascular wall by providing exchange between blood circulation in vessels and tissue fluid and therefore performs a barrier function. Endothelial cells (ECs) in culture are able to maintain the barrier function peculiar to cells of vascular endothelium in vivo. The endothelial monolayer in vitro is a unique model system that allows studying interaction of cytoskeletal and adhesive structures of endotheliocytes from the earliest stages of its formation. In the present work, we described and quantitatively characterized the changes of EC cytoskeleton from the moment of spreading of endotheliocytes on glass and the formation of the first contacts between neighbor cells until formation of a functional confluent monolayer. The main type of intermediate filaments of ECs are vimentin filaments. At different stages of endothelial monolayer formation, disposition of vimentin filaments and their amount do not change essentially, they occupy more than 80% of the cell area. Actin filaments system of endotheliocytes is represented by cortical actin at the cell periphery and by bundles of actin stress fibers organized in parallel. With formation of contacts between cells in native endothelial cells, the number of actin filaments rises and thickness of their bundles increases. With formation of endothelial monolayer, there are also changes in the microtubules system—their number increases at the cell edge. At all stages of EC monolayer formation, the number of microtubules in the region of the already formed intercellular contacts exceeds the number of microtubules in the free lamella region of the cell.     

Changes in the venular epithelium of the mesentery in rats after application of hydrogen peroxide

The change of the venular permeability and endothelial structure was studied with the aid of fluorometry and electron microscopy after application of H2O2. Transmural transfer rate of FITC-albumin++ and permeable fraction of the vessel walls were increased. There were formed the "leaks" and transendothelial canals in the venular endothelium. Numerous local membrane injuries of the endotheliocytes and pericytes, degranulation of the mast cells and destruction of thrombocytes into the vessel lumen were detected.