A role for smad6 in development and homeostasis of the cardiovascular system.

Smad proteins are intracellular mediators of signalling initiated by Tgf-betasuperfamily ligands (Tgf-betas, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-beta superfamily ligands, and downregulate signalling in in vitro assays. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-beta superfamily signalling pathways in vivo.

Monocyte attachment to activated human vascular endothelium in vitro is mediated by leukocyte adhesion molecule-1 (L-selectin) under nonstatic conditions.

The receptors that mediate monocyte adhesion to cytokine-stimulated endothelial monolayers were assessed using a nonstatic (rotating) cell-attachment assay. In this system, leukocyte adhesion molecule-1 (LAM-1) (L-selectin) mediated a major portion (87 +/- 15% at 37 degrees C) of monocyte attachment to activated endothelium. mAb blocking of endothelial leukocyte adhesion molecule-1 (41% inhibition), CD18 (36%), and vascular cell adhesion molecule-1 (25%) function had lesser effects on attachment. These results suggest that LAM-1 may serve an important role in monocyte attachment to endothelium at sites of inflammation.

Tumor dormancy in vivo by prevention of neovascularization.

Dormant solid tumors were produced in vivo by prevention of neovascularization. When small fragments of anaplastic Brown-Pearce carcinoma were implanted directly on the iris in susceptible rabbits, they always vascularized. A characteristic growth pattern, consisting of prevascular, vascular, and late phases, was observed, which terminated with destruction of the eye within 2 wk. The beginning of exponential volume increase was shown to coincide with vascularization of the implant, as demonstrated by perfusion with intravenous fluorescein and by histologic sections. In contrast, implants placed in the anterior chamber, at a distance from the iris, did not become vascularized. After initial growth into spheroids, they remained arrested at a small size comparable to prevascular iris implants, for periods as long as 6 wk. Although dormant in terms of expansion, these avascular tumors contained a population of viable and mitotically active tumor cells. When reimplanted on the iris, vascularization was followed by rapid, invasive growth. These observations suggest that neovascularization is a necessary condition for malignant growth of a solid tumor. When a small mass of tumor cells is prevented from eliciting new vessel ingrowth from surrounding host tissues, population dormancy results. These data suggest that the specific blockade of tumor-induced angiogenesis may be an effective means of controlling neoplastic growth.

Induction and detection of a human endothelial activation antigen in vivo.

We used a murine mAb, H4/18, raised by immunization with IL-1-treated human umbilical vein endothelial cell cultures, to localize an endothelial activation antigen in induced human delayed hypersensitivity reactions (DHR) and in pathological tissues. We used streptococcus varidase to elicit DHR in human skin and we examined sequential skin biopsies with the immunoperoxidase technique. There was no staining for H4/18 binding antigen in normal endothelium of skin and other tissues; strong positive staining, localized to vascular endothelium, was seen at 16 and 23 h but disappeared by 6 d, when the DHR had faded. H4/18 binding antigen, also confined to endothelium, was detected in lymph nodes, skin, and other tissues exhibiting immune/inflammatory reactions. The studies indicate that H4/18 is a useful marker for activated endothelium in vivo and they support the relevance of in vitro studies on inducible endothelial cell functions.

Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells.

Human monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of procoagulant activity in cultured human vascular endothelium. IL-1-induced human umbilical vein endothelial cell procoagulant activity (HEC-PCA) was transiently expressed, manifest in intact cell monolayers, and required protein synthesis. Data obtained with coagulation factor-deficient plasma and a goat anti-human apoprotein III antiserum suggested that most, if not all, of IL-1-induced endothelial cell procoagulant activity is tissue factor-like. IL-1 induction of HEC-PCA may be important in the pathogenesis of intravascular coagulation in a variety of immunological and inflammatory conditions.

Inhibition of E-selectin gene expression by transforming growth factor beta in endothelial cells involves coactivator integration of Smad and nuclear factor kappaB-mediated signals.

Transforming growth factor (TGF)-beta(1) is a pleiotropic cytokine/growth factor that is thought to play a critical role in the modulation of inflammatory events. We demonstrate that exogenous TGF-beta(1) can inhibit the expression of the proinflammatory adhesion molecule, E-selectin, in vascular endothelium exposed to inflammatory stimuli both in vitro and in vivo. This inhibitory effect occurs at the level of transcription of the E-selectin gene and is dependent on the action of Smad proteins, a class of intracellular signaling proteins involved in mediating the cellular effects of TGF-beta(1). Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression. These data thus demonstrate a novel mechanism of interaction between TGF-beta(1)-regulated Smad proteins and NFkappaB proteins regulated by inflammatory stimuli in vascular endothelial cells. This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

Ia expression by vascular endothelium is inducible by activated T cells and by human gamma interferon.

We have used monoclonal antibody binding, measured by radioimmunoassay, fluorescence flow cytometry, and ultrastructural immunocytochemistry, to measure expression of Ia antigens on cultured human umbilical vein endothelial (HUVE) cells. Under standard culture conditions, HUVE cells do not express Ia antigens. However, treatment of primary HUVE cultures with phytohemagglutinin induces the expression of Ia antigens. Every endothelial cell in the culture becomes Ia-positive and endothelial cells appear to synthesize Ia. HLA-A,B is concomitantly increased. The expression of Ia appears to be mediated by T cells because (a) pretreatment of primary HUVE cultures with OKT3 plus complement blocks the action of the lectins but not of medium conditioned by lectin-activated peripheral blood mononuclear cells; (b) co-culture of endothelial cells with allogeneic T cells, in the absence of lectin, also induces endothelial Ia; and (c) human immune (gamma) interferon, produced by Chinese hamster ovary cells transfected with the human gamma interferon gene, directly induces endothelial Ia. During co-culture with lymphocytes, about one-third of the endothelial cells are Ia-positive after 24 h and all of the endothelial cells are Ia-positive by 72 h. Proliferation of allogeneic T cells starts by 96 h and peaks at 144 h. Thus, endothelial Ia appears sufficiently early to be a determinant for the proliferation of allogeneic T cells. Inducible expression of Ia by endothelium may be important both for allograft rejection and for recruitment of circulating T cells into the site of an immune response.

Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules.

Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti-ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo.

Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane-associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.

Factors influencing the expression of stress fibers in vascular endothelial cells in situ.

The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age-matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.

Human vascular endothelial cells in culture. Growth and DNA synthesis.

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [(3)H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [(3)H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [(3)H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.

Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression.

Vascular smooth muscle cells (VSMC) in the rat mesenteric artery show specific immunofluorescent staining with antisera against purified human uterine myosin (ASMM) but not human platelet myosin (APM). However, in primary cultures produced by enzymatic dissociation of this vessel, VSMC stain specifically with both ASMM and APM within 5 h after plating and throughout growth to confluence (4-10 d). In confluent cultures, APM staining remains bright while ASMM staining is reduced in intensity in most cells. In contrast, cellular myosin content, determined by quantitative SDS PAGE, is comparable in confluent and growing cultures. Immunoprecipitation of high salt extracts of cultured VSMC with ASMM and APM yields myosins with the same mobilities on SDS PAGE. When serial, exhaustive precipitations are performed with one antiserum, followed by reprecipitation with the other, myosin in subconfluent and confluent VSMC cultures is exhaustively precipitated by either antiserum, thus indicating complete immunological cross-reactivity. These results might be explained by synthesis of a new myosin isoform reactive with both ASMM and APM. However, the development of APM staining in cultured VSMC did not require protein synthesis. Therefore, it is more likely that the changes in immunofluorescent staining observed in vitro reflect conformational alterations, perhaps related to cytoskeletal rearrangements. These changes in myosin antigenic expression may be relevant to the problem of VSMC phenotypic modulation both in vitro and in vivo.

Functional angiotensin II receptors in cultured vascular smooth muscle cells.

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.

Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.

We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability to associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites. A detergent based extraction and Western blotting procedure demonstrated an association of E-selectin with the insoluble (cytoskeletal) fraction of endothelial monolayers that correlated with adhesion of leukocytes via an E-selectin-dependent mechanism. A mutant form of E-selectin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cell and supported leukocyte attachment (in a nonstatic adhesion assay) in a fashion similar to the native E-selectin molecule, but failed to become associated with the cytoskeletal fraction. To identify the cytoskeletal components that associate with the cytoplasmic domain of E-selectin, paramagnetic beads coated with the adhesion-blocking anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alpha-actinin, vinculin, filamin, paxillin, as well as focal adhesion kinase (FAK), were copurified by this procedure, however talin was not. When a mechanical stress was applied to H18/7-coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoskeletal mechanical linkage. COS-7 cells transfected with the tailless E-selectin failed to show resistance to the twisting stress. Taken together, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin through its cytoplasmic domain, a process which may have important implications for cell-cell signaling as well as mechanical anchoring during leukocyte-endothelial adhesive interactions.

Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, beta 1-integrins, and beta 2-integrins.

Leukocyte interactions with vascular endothelium at sites of inflammation can be dynamically regulated by activation-dependent adhesion molecules. Current models, primarily based on studies with polymorphonuclear leukocytes, suggest the involvement of multiple members of the selectin, integrin, and immunoglobulin gene families, sequentially, in the process of initial attachment (rolling), stable adhesion (arrest), spreading and ultimate diapedesis. In the current study, IL-4-activated human umbilical vein endothelium, which selectively expresses VCAM-1 and an L-selectin ligand but not E-selectin, and appropriate function blocking monoclonal antibodies, were used to study monocyte-endothelial interactions in an in vitro model that mimics microcirculatory flow conditions. In this system, L-selectin mediates monocyte rolling and also facilitates alpha 4 beta 1-integrin-dependent arrest, whereas beta 2-integrins are required for spreading of firmly attached monocytes on the endothelial cell surface but not their arrest. These findings provide the first in vitro evidence for human monocyte rolling on cytokine-activated endothelium, and suggest a sequential requirement for both beta 1- and beta 2-integrin-dependent adhesive mechanisms in monocyte-endothelial interactions.

Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis.

An inducible rabbit endothelial adhesion molecule that is selective for mononuclear leukocytes has been identified. This adhesion protein was expressed on the surface of activated cultured endothelium in two forms, 118 and 98 kilodaltons, the amino-terminal sequence of each being highly homologous to human VCAM-1. In dietary hypercholesterolemic and Watanabe heritable hyperlipidemic rabbit models of atherosclerosis, this adhesion molecule was found to be expressed in a localized fashion by aortic endothelium that overlies early foam cell lesions. This lesion-localized expression suggests a potential endothelium-dependent mechanism for mononuclear leukocyte recruitment during atherogenesis and may provide a molecular marker for early atherosclerosis.

Angiotensin II stimulation of prostaglandin production in cultured human vascular endothelium.

Immunoreactive material resembling prostaglandin E accumulates in the medium of cultured human umbilical vein endothelial cells. Prodcution is inhibited by indomethacin and stimulated by angiotensin II. Prostaglandin secretion by endothelium may be important in platelet-dependent thrombotic phenomena, and in local control of vascular permeability and tone in vivo.

Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins.

Focal adhesion of leukocytes to the blood vessel lining is a key step in inflammation and certain vascular disease processes. Endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell surface glycoprotein expressed by cytokine-activated endothelium, mediates the adhesion of blood neutrophils. A full-length complementary DNA (cDNA) for ELAM-1 has now been isolated by transient expression in COS cells. Cells transfected with the ELAM-1 clone express a surface structure recognized by two ELAM-1 specific monoclonal antibodies (H4/18 and H18/7) and support the adhesion of isolated human neutrophils and the promyelocytic cell line HL-60. Expression of ELAM-1 transcripts in cultured human endothelial cells is induced by cytokines, reaching a maximum at 2 to 4 hours and decaying by 24 hours; cell surface expression of ELAM-1 protein parallels that of the mRNA. The primary sequence of ELAM-1 predicts an amino-terminal lectin-like domain, an EGF domain, and six tandem repetitive motifs (about 60 amino acids each) related to those found in complement regulatory proteins. A similar domain structure is also found in the MEL-14 lymphocyte cell surface homing receptor, and in granule-membrane protein 140, a membrane glycoprotein of platelet and endothelial secretory granules that can be rapidly mobilized (less than 5 minutes) to the cell surface by thrombin and other stimuli. Thus, ELAM-1 may be a member of a nascent gene family of cell surface molecules involved in the regulation of inflammatory and immunological events at the interface of vessel wall and blood.

Vasoconstriction: a new activity for platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.

Endothelial interleukin-8: a novel inhibitor of leukocyte-endothelial interactions.

Certain inflammatory stimuli render cultured human vascular endothelial cells hyperadhesive for neutrophils. This state is transient and reversible, in part because activated endothelial cells secrete a leukocyte adhesion inhibitor (LAI). LAI was identified as endothelial interleukin-8 (IL-8), the predominant species of which is an extended amino-terminal IL-8 variant. At nanomolar concentrations, purified endothelial IL-8 and recombinant human IL-8 inhibit neutrophil adhesion to cytokine-activated endothelial monolayers and protect these monolayers from neutrophil-mediated damage. These findings suggest that endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood.