Luminal Endothelial Surface Research Articles

Leukocyte – endothelial interactions in inflammation

At sites of inflammation, infection or vascular injury local proinflammatory or pathogen-derived stimuli render the luminal vascular endothelial surface attractive for leukocytes. This innate immunity response consists of a well-defined and regulated multi-step cascade involving consecutive steps of adhesive interactions between the leukocytes and the endothelium. During the initial contact with the activated endothelium leukocytes roll along the endothelium via a loose bond which is mediated by selectins. Subsequently, leukocytes are activated by chemokines presented on the luminal endothelial surface, which results in the activation of leukocyte integrins and the firm leukocyte arrest on the endothelium. After their firm adhesion, leukocytes make use of two transmigration processes to pass the endothelial barrier, the transcellular route through the endothelial cell body or the paracellular route through the endothelial junctions. In addition, further circulating cells, such as platelets arrive early at sites of inflammation contributing to both coagulation and to the immune response in parts by facilitating leukocyte–endothelial interactions. Platelets have thereby been implicated in several inflammatory pathologies. This review summarizes the major mechanisms and molecules involved in leukocyte–endothelial and leukocyte-platelet interactions in inflammation.

Vaccination with xenogeneic tumor endothelial proteins isolated in situ inhibits tumor angiogenesis and spontaneous metastasis

Angiogenesis is critical for tumor growth and metastasis. Tumor tissues induce the expression of angiogenesis-associated proteins on endothelial surface that can be targeted for tumor immunotherapy. In our study, the rat tumor endothelial proteins (EP) were isolated in situ via biotinylation of tumor vascular endothelial luminal surface followed by streptavidin affinity chromatography. The isolated tumor EP contained numerous up-regulated angiogenesis-associated endothelial proteins. The administration of these tumor EP as a vaccine to mice reduced the microvessel density in subcutaneous primary LLC tumors, delayed spontaneous LLC tumor metastasis and prolonged post-surgery life span. T lymphocytes from tumor EP-vaccinated mice lysed human umbilical vascular endothelial cells, but not tumor cells in vitro, in a dose-dependent manner. Furthermore, adoptive transfer of antitumor EP antibodies in vivo targeted to tumor endothelium and inhibited spontaneous LLC tumor metastasis. This study provides a successful preclinical exploration of the active immunotherapy for tumor by targeting tumor angiogenesis.

Reduction in endothelial glycocalyx thickness after hemorrhage

Although some evidence suggests that ischemia induces changes in the endothelial glycocalyx (EG), little information is available about the EG following hemorrhagic hypotension (HH). We have employed video analysis to obtain preliminary data on EG changes following hemorrhage. Using intravital videomicroscopy, we studied the spinotrapezius muscle of anesthetized rats subjected to HH (40% blood vol.) for over 1 h. The analog video camera signal was digitized using standard software to DV‐AVI format. About 100 frames of several video clips (3‐5 s each) were processed using specially designed software to derive dimensions of the endothelial glycocalyx from the gap between erythrocytes and the luminal endothelial surface. Frequency distributions of the EG thickness during baseline and during HH showed a shift to lower values following hemorrhage, corresponding to a reduction in EG thickness of nearly 50 % (1.1 µm [range: 0.4 ‐ 4.3 µm] vs 0.6 µm [range: 0.3 ‐ 1.3 µm]). These changes occurred despite similar distributions of anatomic diameters (baseline range: 9 ‐ 24 µm; HH: 10 ‐ 21 µm). The findings suggest that the analysis of EG changes using in vivo video recordings can be used to investigate changes following HH and provide preliminary support for our hypothesis that these low flow states cause reductions in the endothelial surface layer thickness.SupportNIH, NWO, NHS, (HV: NHF grant 2005T037).

Polarized response of endothelial cells to invasion byAspergillus fumigatus

Hyphal invasion of blood vessels is a prominent feature of invasive aspergillosis. During invasive pulmonary aspergillosis, Aspergillus fumigatus hyphae invade the abluminal endothelial cell surface, whereas they invade the luminal endothelial cell surface during haematogenous dissemination. We investigated the endothelial cell response to abluminal and luminal infection with A. fumigatus hyphae in vitro. We found that these hyphae invaded the abluminal endothelial cell surface without inducing the formation of endothelial cell pseudopods. Also, the internalized hyphae were surrounded by a loose network of microfilaments. In contrast, A. fumigatus hyphae invaded the luminal endothelial cell surface by inducing by the formation of endothelial cell pseudopods. These endocytosed hyphae were surrounded by a tight network of microfilaments. Abluminal infection induced greater E-selectin, IL-8, tissue factor and TNF-alpha gene expression, but less endothelial cell damage than did luminal infection. Endothelial cell stimulation by infection of either surface was mediated by endothelial cell-derived TNF-alpha, and was not influenced by gliotoxin secreted by A. fumigatus. These differences in the endothelial cell response to abluminal versus luminal infection may contribute to differences in the pathogenesis of invasive versus haematogenously disseminated aspergillosis.

Subcellular localization of transporters along the rat blood–brain barrier and blood–cerebral-spinal fluid barrier by in vivo biotinylation

Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood–brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal–lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal–lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood–brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

The “mode” of lymphocyte extravasation through HEV of Peyer's patches and its role in normal homing and inflammation

The mode of lymphocyte transendothelial migration in the postcapillary high endothelial venules (HEVs) of Peyer's patches during normal homing and acute inflammation in the guinea pig was studied. It is common opinion that the lymphocyte transendothelial passage from the blood stream into the extravasal lymphoid tissue calls for a multistep process of endothelial and lymphocyte molecules favoring tethering, rolling, activation, arrest and its firm adhesion to the endothelial luminal surface. Ultrastructural serial pictures and the three-dimensional reconstruction of HEVs with lymphocytes during different moments of their transmigration through the endothelial wall enabled us to demonstrate in vivo the morphological modality of their extravasation in lymphoid tissue. The latter is accomplished by means of an intraendothelial canalicular formation (6.8–7.2 μm long and 2.1–2.2 μm in diameter), whose creation depends on the particular behavior of adjacent endothelial cells, without compromising the interendothelial contacts. This new canalicular pathway of lymphocyte extravasation, particularly selective for the B cell, does not permit confirmation of the dogmas of the transcellular and paracellular (open interendothelial junctions) modes that have prevailed in recent decades. The lack of knowledge regarding the molecular bases that would induce constitution of this intraendothelial canalicular formation is a critical point for stimulating future interdisciplinary research aimed at developing strategies for modulating normal lymphocyte homing and in inflammation.

Effects of aging and HIF‐1α deficiency on permeability of hippocampal vessels

We examined age-related changes in the blood-brain barrier (BBB) of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha) deficient mice, which showed hydrocephalus with neuronal cell loss, to investigate an effect of neural cell-specific HIF-1alpha deficiency or hydrocephalus on vascular function. Vascular permeability of horseradish peroxidase (HRP) and binding of cationized ferritin (CF) particles to the endothelial cell luminal surface, as a marker of glycocalyx, were investigated. The thickness of CF-labeled glycocalyx was significantly decreased in the cortex in mutant mice compared with that of control mice, although it was not paralleled by increased vascular permeability. In addition, strong staining for HRP was seen around vessels located along the hippocampal fissure in 24-month-old mutant mice. The reaction product of HRP appeared in an increasing number of the endothelial cell abluminal vesicles and within the thickened basal lamina of arterioles in the hippocampus, showing increased vascular permeability. There were no leaky vessels in 10-week-old mutant mice or 10-week-old and 24-month-old control mice. These findings suggest the necessity of two factors, aging and hydrocephalus, for BBB dysfunction in HIF-1alpha deficient mice.

Screening phage display libraries for organ-specific vascular immunotargeting in vivo

The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs in vivo. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream. To obviate liver uptake of intravenously injected phage, we convert the phage-displayed antibodies into scFv-Fc fusion proteins, which then are able to rapidly target select organ(s) in vivo as visualized directly by gamma-scintigraphic whole-body imaging. Mass spectrometry helps identify the antigen targets. This comprehensive strategy provides new promise for harnessing the power of phage display for mapping vascular endothelia natively in tissue and for achieving vascular targeting of specific tissues in vivo.

Charge modification of the endothelial surface layer modulates the permeability barrier of isolated rat mesenteric small arteries

We hypothesized that modulation of the effective charge density of the endothelial surface layer (ESL) results in altered arterial barrier properties to transport of anionic solutes. Rat mesenteric small arteries (diameter approximately 190 microm) were isolated, cannulated, perfused, and superfused with MOPS-buffered physiological salt solutions. MOPS-solutions were of normal ionic strength (162 mM, MOPS), low ionic strength (81 mM, LO-MOPS), or high ionic strength (323 mM, HI-MOPS), to modulate ESL charge density (normal, high, or low ESL charge, respectively). Osmolarity of MOPS, LO-MOPS, and HI-MOPS was kept constant at 297 mosmol/l, using additional glucose when necessary. Perfusate solutions were supplemented with 1% BSA. Arteries were cannulated with a double-barreled theta-pipet on the inlet side and a regular pipet on the outlet side. After infusion of FITC-labeled dextran of 50 kDa (FITC-Delta50) and the endothelial membrane dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, the dynamics of arterial dye filling were determined with confocal microscopy. ESL thickness, as determined from the initial exclusion zone for FITC-Delta50 on the luminal endothelial surface, was 6.3 +/- 1.4 microm for LO-MOPS, 2.7 +/- 1.0 microm for MOPS, and 1.1 +/- 1.3 microm for HI-MOPS. At low ionic strength, FITC-Delta50 permeated into the ESL with a total ESL permeation time (tauESL) of 26 min, and at normal ionic strength with a tauESL of 20 min. No apparent exclusion of FITC-Delta50 from the ESL could be observed at high ionic strength. In conclusion, we demonstrate that the modulation of solvent ionic strength influences the thickness and barrier properties of the ESL.

Manganese Distribution Across the Blood–Brain Barrier III: The Divalent Metal Transporter-1 is not the Major Mechanism Mediating Brain Manganese Uptake

Manganese (Mn) is essential for and toxic to the brain. Brain Mn uptake utilizes both diffusion and transporter-mediated pathways. The divalent metal transporter-1 (DMT-1) has been suggested to mediate brain Mn uptake. The b/ b Belgrade rat does not express significant amounts of functional DMT-1. In the present work, brain influx transfer coefficients of 54 Mn ion and 54 Mn transferrin (Mn Tf) were determined in b/ b and +/ b Belgrade and Wistar rats using the in situ brain perfusion technique. Brain Mn uptake was not significantly different among the three rat strains for either Mn species. We hypothesized that Mn may enter brain endothelial cells by a DMT-1-independent process but not be able to distribute across those cells into brain tissue due to the absence of DMT-1 activity. To test this hypothesis the brain capillary endothelial cells were isolated from b/ b and +/ b Belgrade rats and Wistar rats after in situ brain perfusion. Some animals received cerebrovascular washout after in situ brain perfusion to ascertain any affect of genotype on 54 Mn adsorption to the endothelial cell luminal surface. Less than 30% of the brain 54 Mn after 54 Mn ion or 54 Mn Tf perfusion remained associated with endothelial cells, suggesting the majority had distributed into brain extracellular fluid (ECF) and/or brain cells. Mn appears to distribute across the rat blood–brain barrier (BBB) into the brain by one or more carrier-mediated processes other than the DMT-1.

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro.

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.

ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface: Presented in part as posters at the American Thoracic Society (ATS) Meeting, May 5-10, 2000, Toronto, ON, Canada.

AbstractDrug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. 125I-labeled anti-ICAM and a reporter enzyme (β-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.

Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide

The vascular endothelium acutely autoregulates blood flow in vivo in part through unknown mechanosensing mechanisms. Here, we report the discovery of a new acute mechanotransduction pathway. Hemodynamic stressors from increased vascular flow and pressure in situ rapidly and transiently induce the activity of neutral sphingomyelinase but not that acid sphingomyelinase in a time- and flow rate-dependent manner, followed by the generation of ceramides. This acute mechanoactivation occurs directly at the luminal endothelial cell surface primarily in caveolae enriched in sphingomyelin and neutral sphingomyelinase, but not acid sphingomyelinase. Scyphostatin, which specifically blocks neutral but not acid sphingomyelinase, inhibits mechano-induced neutral sphingomyelinase activity as well as downstream activation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) by increased flow in situ. We postulate a novel physiological function for neutral sphingomyelinase as a new mechanosensor initiating the ERK cascade and possibly other mechanotransduction pathways.

Coronary flow-induced inotropism is modulated by binding of dextrans to the endothelial luminal surface

In isolated perfused guinea pig hearts, coronary flow causes a positive inotropic effect [positive coronary flow-induced effect (+CFIE)] that could be altered by dextrans (Dx) in the coronary perfusion solution. To test this possibility, Dx of 20, 40, 70, and 500 kDa were infused and found to modulate +CFIE; however, when Dx infusion was terminated, the effect persisted, i.e., was irreversible/nonwashable, suggesting that Dx may bind to luminal endothelial lectinic structures. This hypothesis was tested when Dx [with fluorescent traces (D*)] bound to the vessel wall was hydrolyzed by dextranase infusion and washout of D* fragments completely reverted the +CFIE, and it was found that bound D* to be displaced by free Dx required concentrations 50-100 times that used during binding. In addition, dose-response curves for Dx on +CFIE show that the higher the Dx molecular mass, the lesser the concentration required to have an effect. Because a large Dx molecule has a greater number polymeric glucose branches, it can bind to a larger number of endothelial lectinic sites, requiring a lower concentration to affect +CFIE. Our results suggest that luminal endothelial lectinic structures are part of the flow-sensing assembly.

Synthesis of sulfated proteoglycans by bovine glomerular endothelial cells in culture.

It has been suggested that proteinuria is caused by alterations of the charge selectivity of the basement membrane and/or the epithelial cell layer (podocytes). However, recent findings suggest that the endothelial luminal surface coat, consisting of proteoglycans with their connected glycosaminoglycan (GAG) branches and glycoproteins, may contribute to the permselectivity. Therefore, we wanted to investigate the effects on endothelial GAG synthesis during normal and pathological conditions. We treated glomerular endothelial cell cultures with puromycin aminonucleoside (PAN, a nephrosis-inducing agent) or interleukin-1beta (IL-1beta) for a total of 72 h and compared the metabolic turnover and incorporation of [(35)S]sulfate during the last 2 days. In control cultures, the GAG content in the media supernatants increased 66 +/- 6% (mean +/- SE) between 12 and 42 h of incubation with radioactivity (P < 0.01, n = 8). The content of (35)S-labeled GAGs in the media was reduced by 31 +/- 1% by PAN (P < 0.001, n = 8) and increased by 141 +/- 15% by 10 U/ml IL-1beta (P < 0.01, n = 8). Treatment with enzymes revealed a dominance of heparan, chondroitin, and dermatan sulfate GAGs. Thus the glomerular endothelial cell production of GAGs was increased by IL-1beta and reduced by PAN. Therefore, it is conceivable that certain nephrotic conditions may be due to endothelial dysfunction, rather than other renal causes.

Sole activation of three luminal adenosine receptor subtypes in different parts of coronary vasculature.

In isolated guinea pig hearts saline perfused at constant flow, adenosine A(1), A(2A), and A(3) (A(x)) agonists covalently bound to a large polymer (Pol; 2,000 kDa) were intracoronarily administered, and three effects were studied: dromotropic, vascular and inotropic. The rank order of potencies were the following: dromotropic (Pol-A(2A)Pol-A(1)>Pol-A(3)) and vascular and inotropic (Pol-A(2A)> or =Pol-A(1)Pol-A(3)), where the rank order of potency for Pol-A(x) depends on the part of the coronary vascular network involved; i.e., there is a vascular heterogeneity. The large size of Pol-A(x) prevents extravascular diffusion and causes it to act solely in the endothelial luminal surface. This implies their cardiac effects are due to endothelial mediators. Inhibition of nitric oxide (NO) and prostaglandin (PG) synthesis with N(G)-nitro-l-arginine methyl ester and indomethacin, respectively, show that the three cardiac effects of Pol-A(1) were mediated by NO and PG, whereas for Pol-A(2A) and Pol-A(3) the mediator was mainly NO but not PG. These results suggest that if Pol-A(x) activated the corresponding endothelial A(x)-receptor subtype, a different mediator would be produced.

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.

Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections.

The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors. Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake. Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

The Breast Cancer β4 Integrin and Endothelial Human CLCA2 Mediate Lung Metastasis

Adhesion of blood-borne cancer cells to the endothelium is a critical determinant of organ-specific metastasis. Here we show that colonization of the lungs by human breast cancer cells is correlated with cell surface expression of the alpha(6)beta(4) integrin and adhesion to human CLCA2 (hCLCA2), a Ca(2+)-sensitive chloride channel protein that is expressed on the endothelial cell luminal surface of pulmonary arteries, arterioles, and venules. Tumor cell adhesion to endothelial hCLCA2 is mediated by the beta(4) integrin, establishing for the first time a cell-cell adhesion property for this integrin that involves an entirely new adhesion partner. This adhesion is augmented by an increased surface expression of the alpha(6)beta(4) integrin in breast cancer cells selected in vivo for enhanced lung colonization but abolished by the specific cleavage of the beta(4) integrin with matrilysin. beta(4) integrin/hCLCA2 adhesion-blocking antibodies directed against either of the two interacting adhesion molecules inhibit lung colonization, while overexpression of the beta(4) integrin in a model murine tumor cell line of modest lung colonization potential significantly increases the lung metastatic performance. Our data clearly show that the beta(4)/hCLCA2 adhesion is critical for lung metastasis, yet expression of the beta(4) integrin in many benign breast tumors shows that this integrin is insufficient to bestow metastatic competence on cells that lack invasiveness and other established properties of metastatic cells.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis.

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAF.