Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme

The renal endothelium has been debated as arising from resident hemangioblast precursors that transdifferentiate from the nephrogenic mesenchyme (vasculogenesis) and/or from invading vessels (angiogenesis). While the Foxd1-positive renal cortical stroma has been shown to differentiate into cells that support the vasculature in the kidney (including vascular smooth muscle and pericytes) it has not been considered as a source of endothelial cell progenitors. In addition, it is unclear if Foxd1-positive mesenchymal cells in other organs such as the lung have the potential to form endothelium. This study examines the potential for Foxd1-positive cells of the kidney and lung to give rise to endothelial progenitors. We utilized immunofluorescence (IF) and fluorescence-activated cell sorting (FACS) to co-label Foxd1-expressing cells (including permanently lineage-tagged cells) with endothelial markers in embryonic and postnatal mice. We also cultured FACsorted Foxd1-positive cells, performed in vitro endothelial cell tubulogenesis assays and examined for endocytosis of acetylated low-density lipoprotein (Ac-LDL), a functional assay for endothelial cells. Immunofluorescence and FACS revealed that a subset of Foxd1-positive cells from kidney and lung co-expressed endothelial cell markers throughout embryogenesis. In vitro, cultured embryonic Foxd1-positive cells were able to differentiate into tubular networks that expressed endothelial cell markers and were able to endocytose Ac-LDL. IF and FACS in both the kidney and lung revealed that lineage-tagged Foxd1-positive cells gave rise to a significant portion of the endothelium in postnatal mice. In the kidney, the stromal-derived cells gave rise to a portion of the peritubular capillary endothelium, but not of the glomerular or large vessel endothelium. These findings reveal the heterogeneity of endothelial cell lineages; moreover, Foxd1-positive mesenchymal cells of the developing kidney and lung are a source of endothelial progenitors that are likely critical to patterning the vasculature.     

Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung

To study the biochemical characteristics of endothelium in vivo, we radioiodinated endothelial membrane proteins of the perfused rabbit lung using a water soluble analog of the Bolton-Hunter reagent, 125I-sulfosuccinimidyl (hydroxyphenyl) propionate (125I-s-SHPP). This technique led to a 10-fold increase in specific activity of radioiodinated lung membrane protein compared with our previously reported method using lactoperoxidase and glucose oxidase-catalyzed radioiodination. Tissue autoradiography confirmed that radioiodination was largely confined to the endothelium. Perfusion pressure, wet-to-dry weight ratios, and the morphological appearance of the lungs were within normal limits, indicating that the procedure does not cause apparent lung injury. Lectin binding to a crude membrane fraction of 125I-s-SHPP labeled lung led to isolation of several putative endothelial membrane proteins. Immunoprecipitation studies with appropriate antibodies enabled the identification of radioiodinated angiotensin-converting enzyme and beta 2-microglobulin associated major histocompatibility complex class I molecules in the membrane fraction. This technique will be useful for studying biochemical responses of the endothelium in vivo to a variety of pharmacological and physiology stimuli.     

Proteomic identification of endothelial proteins isolated in situ from atherosclerotic aorta via systemic perfusion

The functional and structural alterations of vascular endothelium contribute to the initiation, progression, and complications of atherosclerotic plaque formation, but limited information is known about the molecular composition and pathways underlying pathological changes during atherosclerosis. We have developed an affinity proteomic strategy for in situ isolation and differential mapping of vascular endothelial proteins in normal and atherosclerotic aorta tissues. The selective labeling was carried out by perfusion of the blood vessels with an active biotin reagent for covalent modification of accessible vascular endothelial proteins. The biotinylated proteins were then enriched by streptavidin affinity chromatography, separated by SDS-PAGE, and subsequently characterized by LC-MS/MS. The described procedure led to the identification of 454 distinct proteins in normal and atherosclerotic aorta tissues. A majority of the proteins are plasma membrane associated and extracellular matrix proteins, and 81 showed altered expressions in atherosclerotic aorta tissue. The differentially expressed proteins are involved in immune and inflammatory responses, cell adhesion, and lipid metabolism. The method provides a new avenue for investigating the endothelial dysfunction and development of atherosclerosis.     

The shear stress of it all: the cell membrane and mechanochemical transduction

As the inner lining of the vessel wall, vascular endothelial cells are poised to act as a signal transduction interface between haemodynamic forces and the underlying vascular smooth-muscle cells. Detailed analyses of fluid mechanics in atherosclerosis-susceptible regions of the vasculature reveal a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow recirculation. Conversely, steady shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. The molecular basis of shear-induced mechanochemical signal transduction and the endothelium's ability to discriminate between flow profiles remains largely unclear. Given that fluid shear stress does not involve a traditional receptor/ligand interaction, identification of the molecule(s) responsible for sensing fluid flow and mechanical force discrimination has been difficult. This review will provide an overview of the haemodynamic forces experienced by the vascular endothelium and its role in localizing atherosclerotic lesions within specific regions of the vasculature. Also reviewed are several recent lines of evidence suggesting that both changes in membrane microviscosity linked to heterotrimeric G proteins, and the transmission of tension across the cell membrane to the cell-cell junction where known shear-sensitive proteins are localized, may serve as the primary force-sensing elements of the cell.     

Identification of tumour-induced changes in endothelial cell surface protein expression: an in vitro model

The selective destruction of the supporting vasculature of tumours has been proposed as a means of therapy. Fundamental to this approach is the identification of suitable targets on tumour-endothelium. To detect proteins that may be up-regulated on the luminal (apical) surface of tumour-associated endothelium confluent endothelial cells were examined following incubation with tumour cell conditioned medium (TCM) from, or co-culture with, a range of breast carcinoma and small cell lung carcinoma (SCLC) cell lines. Exposed endothelial membrane proteins were labelled with sulpho-NHS-biotin and detected by enhanced chemiluminescence following two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blotting. TCM induced varying levels of proliferative activity in endothelial cells; generally breast TCM contained greater mitogenic activity than SCLC TCM. Exposure of human breast and lung microvascular, and umbilical vein endothelial cells to soluble tumour cell factors from several breast cancer and SCLC cells lines produced similar changes in luminal protein profiles: Breast cancer cells and in particular the MDA-MB-231 cell line induced the most pronounced changes. The expression of six proteins was altered consistently on endothelial cells stimulated with soluble tumour cell factors. However, similar changes were observed following incubation with ECGS suggesting that they were related to endothelial cell proliferation per se. As these proteins were altered in breast and lung microvascular, and umbilical vein endothelial cells stimulated by a variety of breast cancer and SCLC cell lines they support the potentially broad applicability of anti-vascular approaches targeted at the endothelium.     

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions

Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.     

In situ fluorescence labeling of sheep lung microvascular endothelium

Summary Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.     

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.

Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.     

Thrombomodulin is preferentially expressed in Balb/c lung microvessels.

Previously, two rat monoclonal antibodies where developed which bind distinct epitopes on a murine glycoprotein, P112, which is expressed primarily in lung capillary endothelium. In this paper we show that P112 is identical to the endothelial anticoagulant protein, thrombomodulin (TM). Several lines of evidence support this conclusion. First, amino acid analysis of P112 shows a high degree of homology to TM, and both molecules exhibit the same mobility in gel electrophoresis. Second, P112 and TM share reactivity for two different monoclonal antibodies. Third, purified P112, like TM, acts as a cofactor for protein C activation. Finally, two cDNA clones identified with P112 polyclonal antiserum contain sequence identity with the known TM cDNA sequence. Quantitative analysis of TM (P112) expression using a two-site monoclonal antibody assay demonstrates that significantly higher levels of TM are found in lung in comparison with other highly vascularized organs, i.e. the kidney and liver. Quantitative Northern blot data coincides with the two-site assay data and demonstrates that the high level of TM expression in lung is not due to preferential binding of the monoclonal antibodies to lung TM but rather to increased production of TM mRNA in the lung relative to other highly vascularized organs. It is suggested that expression of TM is highest in cells from continuous endothelium.     

Endothelial transcytotic machinery involves supramolecular protein-lipid complexes

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules > 20 A and that transcytosis is an N-ethylmaleimide-sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein-lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.     

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Anti-Inflammatory Effect of Targeted Delivery of SOD to Endothelium: Mechanism,Synergism with NO Donors and Protective Effects In Vitro and In Vivo

Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation.     

Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype

Vascular endothelium, the cellular monolayer lining the entire cardiovascular system, is exposed to a variety of biochemical and biomechanical stimuli. Fluid shear stresses generated by blood flow in the vasculature can profoundly influence the phenotype of the endothelium by regulating the activity of certain flow-sensitive proteins (for example, enzymes), as well as by modulating gene expression. The finding that specific fluid mechanical forces can alter endothelial structure and function has provided a framework for a mechanistic understanding of flow-dependent processes, ranging from vascular remodeling in response to hemodynamic changes, to the initiation and localization of chronic vascular diseases such as atherosclerosis.     

Productive dengue virus infection of human endothelial cells is directed by heparan sulfate-containing proteoglycan receptors

Dengue virus causes leakage of the vascular endothelium, resulting in dengue hemorrhagic fever and dengue shock syndrome. The endothelial cell lining of the vasculature regulates capillary permeability and is altered by immune and chemokine responses which affect fluid barrier functions of the endothelium. Our findings indicate that human endothelial cells are highly susceptible to infection by dengue virus (type 4). We found that dengue virus productively infects ～80% of primary human endothelial cells, resulting in the rapid release of ～10(5) virions 1 day postinfection. Analysis of potential inhibitors of dengue virus entry demonstrated that antibodies and ligands to integrins and cellular receptors were unable to inhibit dengue virus infection of endothelial cells. In contrast, pretreating cells with heparin or heparan sulfate resulted in a 60 to 80% reduction in dengue virus-infected cells, and pretreatment of endothelial cells with heparinase III or protease reduced dengue infectivity by >80%. Dengue virus bound specifically to resin immobilized heparin, and binding was competitively inhibited by excess heparin but not other ligands. Collectively, these findings suggest that dengue virus specifically attaches to heparan sulfate-containing proteoglycan receptors on endothelial cells. Following attachment to human endothelial cell receptors, dengue virus causes a highly productive infection that has the potential to increase viral dissemination and viremia. This provides the potential for dengue virus-infected endothelial cells to directly alter barrier functions of the endothelium, contribute to enhancement of immune cell activation, and serve as potential targets of immune responses which play a central role in dengue pathogenesis.     

Synthesis by cultured human umbilical vein endothelial cells of two proteins structurally and immunologically related to platelet membrane glycoproteins IIb and IIIa

Human platelets participate in a number of adhesive interactions, including binding to exposed subendothelium after vascular injury, and platelet-platelet cohesion to form large aggregates. Platelet membrane glycoproteins (GP) IIb and IIIa constitute a receptor for fibrinogen that, together with fibrinogen and calcium, is largely responsible for mediating the formation of the primary hemostatic plug. Using highly specific polyclonal and monoclonal antibodies as probes, we could detect the presence of both of these glycoproteins in cultured human umbilical vein endothelial cells. Western-blot analysis showed that the endothelial cell analogues were similar in size to their platelet counterparts, and were present in cells that had been in culture for over 2 mo. Metabolic labeling of endothelium with [35S]methionine demonstrated that both GPIIb and GPIIIa were actively synthesized in culture. Using the technique of crossed immunoelectrophoresis, evidence was obtained that the endothelial cell forms of GPIIb and GPIIIa may exist complexed to one another after solubilization in Triton X-100. The presence of GPIIb-IIIa analogues in cultured endothelial cells may provide an opportunity to examine the structure, function, and synthesis of these two membrane glycoproteins, as well as provide a source of genetic material with which to begin detailed molecular genetic studies.     

In vivo phage display and vascular heterogeneity: implications for targeted medicine

The vascular endothelium expresses differential receptors depending on the functional state and tissue localization of its cells. A method to characterize this receptor heterogeneity with phage display random peptide libraries has been developed. Using this technology, several peptide ligands have been isolated that home to tissue-specific endothelial cell receptors following intravenous administration. Such peptide ligands, or antibodies directed against specific vascular receptors, can be used to target therapeutic compounds or imaging agents to endothelial cells in vitro and in vivo. Recent advances in the field include identification of novel endothelial receptors expressed differentially in normal and pathological conditions and the isolation of peptides or antibody ligands to such receptors in in vitro assays, in animal models and in a human patient. These milestones, which extend the 'functional map' of the vasculature, should lead to clinical applications in diseases such as cancer and other conditions that exhibit distinct vascular characteristics.     

Vascular response in a non-uterine site to implantation-stage embryos in the rat and guinea-pig: in vivo and ultrastructural studies

Summary Preimplantation-stage embryos were transferred to the anterior eye chamber of recipient rats and guinea-pigs. After implantation had occurred the influence of the embryo on the iris vasculature was examined ultrastructurally. In both species, the earliest effect of embryonic implantation was an increased stromal oedema. Under increasing embryonic influence the vascular endothelial cells showed an increased number of projections into the vascular lumen, while in the rat, endothelial projections were also found pushing back into the basement membrane. In the rat, the endothelium became very irregular in thickness prior to complete disintegration and loss during more advanced stages of implantation. Rat embryonic trophoblast was found invading iris vasculature, particularly in areas where the iridial endothelium was partially or completely missing. Other cells in the iris, including the stroma, appeared to be less affected. In the guinea-pig, however, trophoblast cells appeared to be capable of invading the vasculature by displacing endothelial cells that still appeared morphologically normal.     

Computerized analysis of tumor cell interactions with extracellular matrix proteins, peptides, and endothelial cells under laminar flow

Arrest and formation of stable adhesive interactions between circulating cells and the endothelium or exposed subendothelial matrix are important processes in many biological situations. We have developed a highly sensitive hydrodynamic assay that utilizes a parallel-plate flow chamber, video microscopy, and digital image processing to separate and measure the primary arrest and adhesion stabilization of flowing cells. Our data indicate that primary cell contact triggers secondary adhesion stabilization, and the secondary events are likely to be critical to metastasis formation. To study the relationship between tumor cell adhesion stabilization and organ-specific blood-borne metastasis, we investigated the adhesion stabilization of metastatic murine RAW117 large-cell lymphoma cells to the extracellular matrix proteins fibronectin and vitronectin, several Arg-Gly-Asp (RGD) containing peptides, and microvascular endothelial cells from the liver or lung. The highly liver metastatic RAW117-H10 subline showed the fastest stabilization to fibronectin, vitronectin, and RGD peptides. Poorly metastatic RAW117-P cells had stabilization times 3-10 times longer than for RAW117-H10 cells, while the lung- and liver-metastatic RAW117-L17 subline failed to stabilize at all. The adhesion stabilization of the RAW117-H10 cells to the extracellular matrix proteins and RGD peptides was inhibited by anti-beta(3) integrin monoclonal antibodies and RGD peptides. In contrast, the RAW117-L17 subline had the shortest stabilization time to unstimulated microvascular endothelial cells of the lung and hepatic sinusoids, followed by RAW117-H10 cells and RAW117-P cells. Monoclonal antibodies against the beta(3) integrin subunit and RGD peptides did not inhibit adhesion stabilization of RAW117-H10 cells to endothelial cells, suggesting that different metastatic variants of large-cell lymphoma cells use differing mechanisms to adhere to organ-specific endothelial cells. (c) 1996 John Wiley & Sons, Inc.