Membrane type 1-matrix metalloproteinase is activated during migration of human endothelial cells and modulates endothelial motility and matrix remodeling.

Matrix metalloproteinases are thought to play an important role in endothelial cell migration and matrix remodeling. We have used an in vitro wound healing migration model and newly generated anti-membrane type 1-matrix metalloproteinase (MT1-MMP) monoclonal antibodies (mAbs) to characterize the role of MT1-MMP during this process. First, the expression and shedding of MT1-MMP are up-regulated upon induction of migration in endothelial cells, as demonstrated by flow cytometry and Western blot analysis. Furthermore, MT1-MMP is concentrated at discrete areas in migrating endothelial cells, in contrast to the diffuse pattern observed in confluent cells. Interestingly, migration of endothelial cells results in the stimulation of MT1-MMP activity, as shown by its ability to process pro-MMP-2 and to degrade fibrinogen assessed by zymography. Moreover, MT1-MMP-mediated gelatin degradation is enriched at migration sites. mAbs generated against the MT1-MMP catalytic domain are shown to inhibit MT1-MMP enzymatic activity and to impair both phorbol 12-myristate 13-acetate-induced endothelial migration and invasion of collagen and fibrin gels. Furthermore, a reduction in the formation of capillary tubes in Matrigel is also observed when endothelial cells are pretreated with the blocking anti-MT1-MMP mAbs. Altogether, these data demonstrate that MT1-MMP plays an important role during endothelial cell migration, and its activity can modulate endothelial migration, invasion, and formation of capillary tubes during the angiogenic response.     

Surfactant protein D is expressed and modulates inflammatory responses in human coronary artery smooth muscle cells

Surfactant protein D (SP-D) is a constituent of the innate immune system that plays a role in the host defense against lung pathogens and in modulating inflammatory responses. While SP-D has been detected in extrapulmonary tissues, little is known about its expression and function in the vasculature. Immunostaining of human coronary artery tissue sections demonstrated immunoreactive SP-D protein in smooth muscle cells (SMCs) and endothelial cells. SP-D was also detected in isolated human coronary artery SMCs (HCASMCs) by PCR and immunoblot analysis. Treatment of HCASMCs with endotoxin (LPS) stimulated the release of IL-8, a proinflammatory cytokine. This release was inhibited >70% by recombinant SP-D. Overexpression of SP-D by adenoviral-mediated gene transfer in HCASMCs inhibited both LPS- and TNF-alpha-induced IL-8 release. Overexpression of SP-D also enhanced uptake of Chlamydia pneumoniae elementary bodies into HCASMCs while attenuating IL-8 production induced by bacterial exposure. Both LPS and TNF-alpha increased SP-D mRNA levels by five- to eightfold in HCASMCs, suggesting that inflammatory mediators upregulate the expression of SP-D. In conclusion, SP-D is expressed in human coronary arteries and functions as an anti-inflammatory protein in HCASMCs. SP-D may also participate in the host defense against pathogens that invade the vascular wall.     

Serglycin secretion is part of the inflammatory response in activated primary human endothelial cells in vitro

Background

Endothelial cells have important functions in e.g. regulating blood pressure, coagulation and host defense reactions. Serglycin is highly expressed by endothelial cells, but there is limited data on the roles of this proteoglycan in immune reactions.

Methods

Cultured primary human endothelial cells were exposed to proinflammatory agents lipopolysaccharide (LPS) and interleukin 1β (IL-1β). The response in serglycin synthesis, secretion and intracellular localization and effect on the proteoglycan binding chemokines CXCL-1 and CXCL-8 were determined by qRT-PCR, Western blotting, immunocytochemistry, ELISA and serglycin knockdown experiments.

Results

Both LPS and IL-1β increased the synthesis and secretion of serglycin, while only IL-1β increased serglycin mRNA expression. Stimulation increased the number of serglycin containing vesicles, with a greater portion of large vesicles after LPS treatment. Also, increased intracellular and secreted levels of CXCL-1 and CXCL-8 were observed. The increase in CXCL-8 secretion was unchanged in serglycin knockdown cells. However, the increase in CXCL-1 secretion from IL-1β stimulation was reduced 27% in serglycin knockdown cells; while the LPS-induced secretion was not affected. In serglycin expressing cells CXCL-1 positive vesicles were evenly distributed throughout the cytoplasm, while confided to the Golgi region in serglycin knockdown cells. This was the case only for IL-1β stimulated cells. LPS-induced CXCL-1 distribution was unaffected by serglycin expression.

Conclusions

These results suggest that different signaling pathways are involved in regulating secretion of serglycin and partner molecules in activated endothelial cells.

General significance

This knowledge increases our understanding of the roles of serglycin in immune reactions. This article is part of a Special Issue entitled: Matrix-mediated cell behaviour and properties.     

Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1⁺ cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies.     

Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes

We have identified new activating receptors of the Ig superfamily expressed on human myeloid cells, called TREM (triggering receptor expressed on myeloid cells). TREM-1 is selectively expressed on blood neutrophils and a subset of monocytes and is up-regulated by bacterial LPS. Engagement of TREM-1 triggers secretion of IL-8, monocyte chemotactic protein-1, and TNF-alpha and induces neutrophil degranulation. Intracellularly, TREM-1 induces Ca2+ mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C-gamma. To mediate activation, TREM-1 associates with the transmembrane adapter molecule DAP12. Thus, TREM-1 mediates activation of neutrophil and monocytes, and may have a predominant role in inflammatory responses.     

A novel conserved nuclear localization signal is recognized by a group of yeast importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.     

Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.     

Proinflammatory effects of copper deficiency on neutrophils and lung endothelial cells

Dietary copper deficiency increases the accumulation of circulating neutrophils in the rat lung microcirculation. This process includes neutrophil adhesion to, migration along, and emigration though the vascular endothelium. The current study was designed to examine the role of copper in each of these steps. Neutrophils were isolated from rats fed either a copper-adequate (CuA, 6.1 microg Cu/g diet) or copper-deficient diet (CuD, 0.3 microg Cu/g diet) for 4 weeks. First, transient and firm adhesion of neutrophils to P-selectin in a flow chamber showed there were more adhered CuD neutrophils than CuA ones. This effect is probably caused by the increased expression of CD11b that was observed in the current study. Second, the evaluation of neutrophil migration under agarose showed that the CuD neutrophils moved farther than the CuA group in response to IL-8 but not fMLP; this suggests an increased sensitivity to a CD11/CD18-independent signalling pathway. Third, the contractile mechanism of endothelial cells was studied. Elevated F-actin formation in Cu-chelated lung microvascular endothelial cells suggests that neutrophil emigration may be promoted by enhanced cytoskeletal reorganization of the endothelium during copper deficiency. Combined, these results support the theory that dietary copper deficiency has proinflammatory effects on both neutrophils and the microvascular endothelium that promote neutrophil-endothelial interactions.     

ETV-2 activated proliferation of endothelial cells and attenuated acute hindlimb ischemia in mice

Ischemia is the reduction of blood flow to tissues by injury of blood vessels. Depending on the sites of tissues and grade of ischemia, ischemia can cause many serious complications. This study aimed to evaluate the effects of the E-twenty six (ETS) factor Ets variant 2 (ETV2) gene expression in angiogenesis and the effect of ETV2 gene therapy in a mouse model of hindlimb ischemia. The role of ETV2 on endothelial cell proliferation was evaluated in vitro. Knockdown of ETV2 expression was done using short hairpin RNA (shRNA) lentiviral viral particles. The ETV2 viral vector was injected into the skeletal muscles at the ligated and burned sites of the hindlimb and evaluated for its efficacy as a gene therapy modality for ischemia. Vascular regeneration in mice was indirectly evaluated by changes in mouse survival, necrotic grades of the leg, normal blood oxygen saturation level (SpO₂), and blood flow by trypan blue injection assay. Preliminary data showed that ETV2 expression played a role in angiogenesis of endothelial cells. ETV2 overexpression could trigger and stimulate proliferation of skeletal endothelial cells. In vivo knockdown of ETV2 expression inhibited the auto-recovery of ischemic hindlimb, while overexpression of ETV2 helped to rescue leg loss and reduce necrosis, significantly improving angiogenesis in hindlimb ischemia. Our findings demonstrate that ETV2 gene therapy is a potentially effective modality for vascular regeneration.     

Monoclonal antibodies to antigens found on the neutrophils, activated T-lymphocytes and blast cells in human acute leukemia

Two hybridomas secreting mouse cytotoxic monoclonal antibodies to in vivo and in vitro activated human T-lymphocyte and neutrophil surface membrane antigenic determinants have been produced. One of these monoclonal antibodies (Ta/H-2) appeared to be also specifically reactive to blast cells in the majority of non-T-non-B and T acute lymphoblastic leukemia patients.     

The stimulation of the bactericidal activity of human neutrophils by lymphoid cells activated by interleukin-2; the effect of inflammatory cytokines]

Regulation of bactericidal activity of neutrophils (BAN) of healthy volunteer blood donors was studied. Interleukin-2 (IL-2)-activated lymphocytes potentiated BAN more effectively then resting lymphocytes. IL-2-activated mononuclear cells (containing lymphocytes and monocytes/macrophages) decreased neutrophil-potentiating activity when compared with nonactivated mononuclear cells. It was concluded that IL-2-activated monocytes exerted potent suppressive influence upon lymphocytes. Recombinant interleukin-1 beta, tumour necrosis factor-alpha, interferon-gamma acted synergistically with IL-2-activated lymphocytes on BAN when the level of neutrophil bactericidal activity was low.     

Semiconservative and unscheduled DNA synthesis on mammalian cells and its modification by glyoxylic compounds

The interaction of glyoxal and four other glyoxylic compounds with semiconservative DNA synthesis and with unscheduled DNA synthesis induced by 25-G X-rays on TC-SV40 hamster cells has been studied. Both syntheses were evaluated by measuring the incorporation of [3H-methyl]-thymidine into the newly synthetized DNA. The unscheduled DNA synthesis amounts to 4% of semiconservative synthesis. The modification of both syntheses by the glyoxylic compounds was tested using the products at non-toxic concentrations for the cells. All the glyoxals inhibited semiconservative DNA synthesis and potentiated unscheduled DNA synthesis at rather similar levels. These effects have been compared with the radiosensitizing activity of these glyoxals in the same TC-SV40 cells and no relationship could be established.     

Dexamethasone inhibits tumor necrosis factor-alpha-induced expression of macrophage inflammatory protein-2 and adhesion of neutrophils to endothelial cells

Macrophage inflammatory protein-2 (MIP-2) belongs to the C-X-C subfamily of chemokines and appears to play an important role in cytokine-induced inflammatory and immune cell-mediated responses. We found that tumor necrosis factor-alpha (TNF-alpha) time- and dose-dependently increased gene and protein expression of MIP-2 in endothelial cells. Moreover, it was observed that dexamethasone treatment inhibited endothelial cell expression of MIP-2 in response to TNF-alpha stimulation and markedly reduced the number of adherent neutrophils. Moreover, we found that a monoclonal antibody against murine MIP-2 abolished neutrophil adhesion to TNF-alpha-activated endothelial cells. These data demonstrate that TNF-alpha induces expression of MIP-2 in endothelial cells and support the hypothesis that the anti-inflammatory action of dexamethasone may, at least in part, be attributable to an inhibition of MIP-2 induction on cytokine-activated endothelial cells.     

Identification and characterization of carbohydrate molecules in mammalian cells recognized by dengue virus type 2

The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells.     

A novel carbohydrate, differentiation antigen on fucogangliosides of human myeloid cells recognized by monoclonal antibody VIM-2

A mouse monoclonal antibody, VIM-2, specific for human blood cells of myelomonocytic lineage, was found to bind to a series of minor gangliosides isolated from the cells of patients with chronic myelogenous leukemia (Uemura, K., Macher, B.A., DeGregorio, M., Scudder, P., Buehler, J., Knapp, W., and Feizi, T. (1985) Biochim. Biophys. Acta 846, 26-36). TLC immunostaining studies with the VIM-2 antibody of gangliosides from normal human neutrophils, acute myeloid leukemia, and chronic myelogenous leukemia cells showed that the total amount and the ratio of the VIM-2 gangliosides varies among these different myeloid cells and appears to be related to the level of cellular differentiation. Purification of these gangliosides from chronic myelogenous leukemia cells was aided by a sensitive enzyme-linked immunosorbent assay procedure used in conjunction with high performance liquid chromatography. Structures for two of the immunoreactive gangliosides (a ceramide decasaccharide, VIII3NeuAcV3-Fuc-nLc8Cer and a ceramide dodecasaccharide X3-NeuAcVII3Fuc-nLc10Cer) are proposed from negative ion fast atom bombardment mass spectrometry of the native gangliosides, methylation analysis, and the combined use of glycosidase treatment and TLC immunostaining with carbohydrate sequence specific antibodies. The VIM-2 antigen was thus characterized as involving the sialofucooligosaccharide sequence.