MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation.

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf ß1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf ß1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α-Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells.

AIMS: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. METHODS: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. RESULTS: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. CONCLUSIONS: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.

Spatiotemporal expression of chemokines and chemokine receptors in experimental anti-myeloperoxidase antibody-mediated glomerulonephritis.

Myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated necrotizing crescentic glomerulonephritis (NCGN) is characterized by abundant leucocyte infiltration. Chemokines are chemotactic cytokines involved in receptor-mediated recruitment of leucocytes. Our objective was to analyse spatiotemporal gene expression of chemokines and chemokine receptors in anti-MPO-mediated NCGN, to find potential targets for intervening with leucocyte influx. NCGN was induced in mice by co-administration of anti-MPO immunoglobulin (Ig)G and lipopolysaccharide. mRNA expression levels of chemokines and chemokine receptors were analysed in whole kidney lysates as well as in laser microdissected glomeruli and tubulo-interstitial tissue 1 and 7 day(s) after NCGN induction. Several chemokines and chemokine receptors were induced or up-regulated in anti-MPO-mediated NCGN, both on day 1 (chemokines CCL3, 5; CXCL2, 5, 13; receptor CXCR2) and on day 7 (chemokines CCL2, 5, 7, 8, 17, 20; CXCL1, 2, 5, 10; CX(3)CL1; receptors CCR2, 8; CX(3)CR1). The expression levels of most chemokines and receptors were higher in glomeruli than in the tubulo-interstitium. Because of the temporal induction of CXCR2 on day 1, we hypothesized CXCR2 as a potential target for treatment in anti-MPO-induced NCGN. Inhibition of CXCR2 using a goat-anti-CXCR2 serum prior to NCGN induction increased glomerular neutrophil influx but did not affect crescent formation and albuminuria. In conclusion, expression levels of various chemokines and chemokine receptors were increased in anti-MPO NCGN, and expressed particularly in glomeruli. These chemokines and receptors may serve as potential targets for treatment. Inhibition of a single target, CXCR2, did not attenuate anti-MPO NCGN. Combinatorial interventions may be necessary to avoid redundancy.

Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature.

Selective occlusion of tumor vasculature was tested as a therapy for solid tumors in a mouse model. The formation of blood clots (thrombosis) within the tumor vessels was initiated by targeting the cell surface domain of human tissue factor, by means of a bispecific antibody, to an experimentally induced marker on tumor vascular endothelial cells. This truncated form of tissue factor (tTF) had limited ability to initiate thrombosis when free in the circulation, but became an effective and selective thrombogen when targeted to tumor endothelial cells. Intravenous administration of the antibody-tTF complex to mice with large neuroblastomas resulted in complete tumor regressions in 38 percent of the mice.

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

Neoglycoproteins as carriers for antiviral drugs: synthesis and analysis of protein-drug conjugates.

In order to investigate whether neoglycoproteins can potentially act as carriers for targeting of antiviral drugs to certain cell types in the body, various neoglycoproteins were synthesized using thiophosgene-activated p-aminophenyl sugar derivatives. These neoglycoproteins were conjugated with the 5'-monophosphate form of the antiviral drug AZT. For a proper characterization of these preparations, both protein and drug content have to be determined. Comparison of the Lowry and the Bio-Rad protein assays revealed that for both the neoglycoprotein carriers themselves and the AZTMP conjugates, the Lowry assay yielded the most reliable and reproducible results. It was demonstrated that both the reagent used for drug conjugation (ECDI) as well as the introduction of phenyl-sugar groups in the protein interfered with the analysis of bound nucleotide as based on spectral differences between protein and protein-drug conjugate. Therefore, we developed a rapid HPLC system for determination of the drug-protein coupling ratio through acid hydrolysis of the covalently bound nucleotide. With the ECDI-mediated conjugation of 5'-monophosphate drug derivatives to neoglycoproteins, products with molar ratios of drug to protein ranging from 1.2 to 5.6 were obtained. The drug-neoglycoprotein conjugates appeared to be fairly stable during storage, in lyophilized form, at -20 degrees C. The anti-HIV-1 activity of the neoglycoprotein-drug conjugates, as determined in vitro in MT-4 cells, was shown to be dependent on glycosylation of the albumin and also on the kind of sugar present in the neoglycoprotein. The anti-HIV-1 activity of the AZTMP-mannose-albumin conjugate exceeded that of the parent drug by more than 4 times.

Donor brain death reduces survival after transplantation in rat livers preserved for 20 hr.

BACKGROUND: Eighty percent of donor organs come from donors who have suffered brain trauma (brain-dead donors). This unphysiological state alters the hemodynamic and hormonal status of the organ donor. This can cause organ injury, which has been suggested to alter the immunological or inflammatory status of the organ after transplantation, and may lead to increased sensitivity of the organ to preservation/transplantation injury. In this study we asked the question: does brain death cause injury to the liver that decreases successful liver preservation? METHODS: The rat liver transplant model was used to compare survival in rats receiving a liver from a brain-dead donor versus a non-brain-dead donor. Brain death was induced by inflation of a cranially placed balloon catheter. The rats were maintained normotensive with fluid infusion for 6 hr. The livers were flushed with University of Wisconsin (UW) solution and immediately transplanted or cold stored for 20 hr before transplantation. RESULTS: Recipient survival with immediately transplanted livers or those stored for 20 hr was 100% with livers from non-brain-dead donors. However, survival decreased when livers were procured from brain-dead donors. Survival was 75% (6/8) when storage time was 0 hr and 20% (2/10) when the liver was cold stored for 20 hr before transplantation. CONCLUSION: This study shows that brain death induces alterations in the donor liver that make it more sensitive to preservation/reperfusion injury than livers from donors without brain death. The mechanism of injury to the liver caused by brain death is not known. Because most livers used clinically for transplantation come from brain-dead donors, it is possible that poor function of these livers is due to the intrinsic condition of the donor organ, more than the quality of the preservation. Methods to treat the brain-dead donor to improve the quality of the liver may be needed to allow better preservation of the organ and to give better outcome after liver transplantation.

Tumor vasculature targeted therapies: getting the players organized.

Based on their location and central role in solid tumor growth, tumor vascular endothelial cells may present an attractive target for the delivery of therapeutic drugs or cells. The potency of blocking the tumor blood supply in eradicating solid tumors was demonstrated recently in a mouse model of tumor vasculature targeting (Huang et al., Science 275: 547-550, 1997). For clinical application of such strategies, tumor endothelium specific target epitopes need to be identified. Recent studies on angiogenesis have identified angiogenesis-related molecules as potential target epitopes. Among these are vascular endothelial growth factor (VEGF)/VEGF-receptor complex, alpha(v) integrins, and Tie receptor tyrosine kinases. Besides blockade of their signalling cascades leading to inhibition of angiogenesis, these epitopes may also be instrumental in tumor vessel specific delivery of therapeutics. Data on the efficacy of therapeutic modalities aimed at these, mostly heterogeneously distributed tumor endothelial epitopes are scarce, and sophisticated experimentation is required to rationalize the development of new therapeutic strategies. Importantly, only detailed evaluations in cancer patients will provide the blueprint for the development of clinically effective tumor vascular targeted therapies.

Targeting of antiviral drugs to T4-lymphocytes. Anti-HIV activity of neoglycoprotein-AZTMP conjugates in vitro.

The delivery of the anti-HIV agent 3'-azido-3'-deoxythymidine (AZT), in its 5'-monophosphate form, (in) to human T-lymphocyte MT-4 cells in vitro through covalent coupling to neoglycoproteins was investigated. In vivo application of this drug targeting concept may lead to increased efficacy and/or diminished side effects caused by AZT during the treatment of AIDS and ARC patients. The rationale for the design of the neoglycoprotein carriers is based on the existence of sugar recognizing lectins on T-lymphocytes. Using a phenyl-linkage between sugar and Human Serum Albumin (HSA), various mannose-, fucose-, galactose-and glucose-containing neoglycoproteins were synthesized. The intrinsic anti-HIV activity of these neoglycoproteins was tested in vitro in HIV-1 infected MT-4 cells. Only the derivative having 40 moles mannose per mole protein (Man40HSA) shows pronounced anti-HIV-1 activity itself. This effect may be caused by interference of the Man40HSA with the gp120-CD4 mediated virus/MT-4 cell interaction. After conjugation with AZTMP, the mannose- as well as the fucose- and galactose-containing conjugates exhibited a pronounced activity. Conjugates of glucose-HSA and HSA displayed much less activity in spite of the fact that drug loading was considerably higher, compared with the galactose, mannose and fucose derivatives. In the series of mannose-neoglycoproteins, the Man22HSA-AZTMP conjugate was shown to be more than 30 times as active against HIV-1 compared to HSA-AZTMP. Selectivity indices of Man7 and Man22HSA-AZTMP were exceeding the AZT and AZTMP indices, indicating that these conjugates possess a more selective action. Stability experiments indicate that the potent action of the galactose-, mannose- and fucose-HSA-AZTMP conjugates is not due to a complete extracellular hydrolysis of the covalent drug-protein bond. Since Man22HSA has no intrinsic activity in the concentration range used, the antiviral effect is unlikely to be explained by synergism of the neoglycoprotein by a component of the cell membrane and subsequent internalization and release of the drug from the conjugate may play a role.

Bi-specific antibody therapy for the treatment of cancer.

Bi-specific antibodies (BsAbs) combine immune cell activation with tumor cell recognition as a result of which tumor cells are killed by pre-defined effector cells. In this review a brief introduction to monoclonal antibodies will precede a more in-depth presentation of the current status of BsAb therapy for cancer. Target molecules and effector mechanisms aimed at tumor cells or aimed at tumor vasculature, and the application of recombinant DNA technology in the construction of antibodies, will be discussed. The lessons learned from the last decade will be discussed in consideration of the potential future development of BsAbs for cancer therapy.

Antiviral activities of lactoferrin.

Lactoferrin (LF) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to LF. One of the functions of LF is the transport of metals, but LF is also an important component of the non-specific immune system, since LF has antimicrobial properties against bacteria, fungi and several viruses. This review gives an overview of the present knowledge about the antiviral activities and, when possible, the antiviral modes of action of this protein. Lactoferrin displays antiviral activity against both DNA- and RNA-viruses, including rotavirus, respiratory syncytial virus, herpes viruses and HIV. The antiviral effect of LF lies in the early phase of infection. Lactoferrin prevents entry of virus in the host cell, either by blocking cellular receptors, or by direct binding to the virus particles.

Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy.

Laser dissection microscopy was applied to isolate endothelial cells from tumors obtained from mice treated with TNF-alpha. RNA integrity was demonstrated from whole sections and dissected cells after acetone fixation and hematoxylin staining. RT-PCR for GAPDH, CD31, VCAM-1, ICAM-1, and E-selectin was successfully performed on these samples. These data demonstrate the feasibility of analyzing local endothelial cell responses in diseased tissues at the level of gene expression.

Disease-induced drug targeting using novel peptide-ligand albumins.

UNLABELLED: Small therapeutic oligopeptides (two to 12 amino acids), designed for interaction with cytokine and growth factor receptors, unfortunately, are rapidly removed from the body. Efficient glomerular filtration and carrier-mediated membrane transport processes are involved in their clearance. By coupling of such peptides to macromolecules, elimination via these pathways is prevented and exposure to the particular receptors can be largely improved. Some of these constructs undergo receptor-mediated endocytoses and can be used as carriers to deliver associated drugs to various cell types in the body. It has been shown that, in the case of neo-glycoprotein carriers, down-regulation of the receptors aimed at can occur in the diseased state. We therefore designed a new type of polypeptide carrier, homing on receptors that are known to be highly upregulated in the pathological target tissue. For this purpose we designed ligand peptides (minimized proteins) representing the receptor-recognizing domains of PDGF and collagen type VI, aimed at receptors that are highly expressed, particularly on activated hepatic stellate cells (HSC). This myofibroblast-type of cell largely contributes to connective tissue expansion during liver fibrosis. Drug carriers for the stellate cell have not been reported before. METHODS: Cyclic octapeptide moieties (n10--12) with affinity for the two receptors were coupled to HSA (pPB-HSA and pCVI-HSA, respectively). Receptor binding experiments confirmed binding of these ligand peptides to their receptors in vitro. IN VITRO STUDIES: rat HSC were isolated and purified according to standard techniques. The cells were cultured for 2 days (quiescent phenotype) or for 10 days (activated phenotype). Cell cultures were incubated with the carriers and the binding (at 4 degrees C), uptake (at 37 degrees C), and degradation were determined with radioactive and immunohistochemical methods. The results were compared with data obtained with unmodified HSA. IN VIVO STUDIES: the organ distribution of pCVI-HSA and pPB-HSA was determined 10 min after i.v. injection of tracer doses in normal and fibrotic rats, 3 weeks after bile duct ligation. Hepatocellular distribution was scored after double-immunostaining of the liver sections with an antibody against the designated hepatic cell type in combination with anti-HSA IgG. IN VITRO STUDIES: All three carriers preferentially bound to the activated rather than to quiescent HSC. Binding to cells was inhibitable by an excess of unlabelled pCVI-HSA, endocytosis was inhibitable by 2 mM monensin suggestive of lysosomal routing of the proteins, whereas pPB-HSA, at least partly, remained at the cell surface. Degradation products of the carriers were detected extracellularly after incubation with fibrotic rat liver slices during 2-h experiments. IN VIVO STUDIES: 62+/-6% of the dose of pCVI-HSA accumulated in fibrotic livers at 10 min after injection, of which the major part was taken up in HSC. 48+/-9% of pPB-HSA accumulated in fibrotic rat livers and this carrier was also mainly taken up by HSC (5). Similar amounts of both constructs were taken up in normal rat livers, but predominantly in other cell types. The preferential homing to the stellate cells, only in the fibrotic liver is explained by the marked proliferation of this cell type as well as overexpression of the targeted receptors on these cells in the diseased state. CONCLUSIONS: The in vivo results support the in vitro studies showing accumulation of these modified albumins in HSC in fibrotic rat livers and, in particular, in the stellate cells. The results demonstrate the specificity of the stellate cell targeting and imply applicability of pCVI-HSA as carriers for drugs that act intracellularly. In addition, pPB-HSA may be used to deliver drugs that act extracellularly, such as receptor antagonists. This concept may create new opportunities for delivery of conventional drugs that are not effective enough in vivo and/or display serious extrahepatic side-effects. Minimized proteins attached to soluble or particle type of macromolecules represent a novel carrier modality of which selective body distribution is induced by the disease process to be targeted. They can be utilized as receptor antagonists and at the same time can deliver therapeutic agents to the desired site of action (dual targeting).

The use of bispecific antibodies in tumor cell and tumor vasculature directed immunotherapy.

To overcome dose limiting toxicities and to increase efficacy of immunotherapy of cancer, a number of strategies are under development for selectively redirecting effector cells/molecules towards tumor cells. Many of these strategies exploit the specificity of tumor associated antigen recognition by monoclonal antibodies. Using either hybridoma fusion, chemical derivatization or molecular biology technology, antibodies with dual specificity can be constructed. These so called biospecific antibodies (BsAbs) have been used to redirect the cytolytic activity of a variety of immune effector cells such as cytotoxic T lymphocytes, natural killer cells, neutrophils and monocytes/macrophages to tumor cells. Local administration of BsAbs, either alone or in combination with autologous effector cells, is highly effective in eradicating tumor cells. In contrast, systemic application of BsAb at present is only suitable for adjuvant treatment of minimal residual disease due to poor tumor cell accessibility. As an alternative, angiogenesis related determinants on tumor blood vessels can be exploited for the selective delivery of effector cells/molecules apart from being used to inhibit angiogenesis. Important advantages of this strategy is that the endothelial cell associated target epitope(s) are easy accessible. The dependence of tumor growth on the tumor's blood supply also renders tumor endothelial cells an attractive target for therapy. Although still in its infancy, attacking the tumor's blood supply for example by delivering coagulation factors or toxins, or by BsAb directed immunotherapies holds great promise for antineoplastic therapy.

The effect of surgical wounding on tumour development.

For more than a century, a role for wound healing in the outgrowth of tumours has been implied based on observations in both experimental and clinical studies. Wound healing can be divided into stages of inflammatory, proliferative, repair and remodelling processes. Through proper regulation of activation of epithelial, endothelial and inflammatory cells, platelets and fibroblasts, and the production of growth factors, wounds heal and the various cell types resume their normal function. In tumour growth, similar processes of cell activation and growth factor production are observed. These processes are, however, differently regulated leading to ongoing cellular activation. In recent years, growth factors such as EGF, TGF-alpha and TGF-beta, bFGF, IGF I and II, and PDGF have been identified to play a role in the different stages of wound healing. In addition, some of these factors have now been identified as also being involved in the outgrowth of tumours. In this review, cell types involved in wound healing and tumour growth, as well as the growth factors and cytokines they produce and the role of the extracellular matrix, extensively present in both conditions, are being discussed. A better understanding of the time interval during which the sequelae of events in wound healing occur in relation to the time interval of tumour recurrence may be the basis for defining new therapeutic strategies that can interfere with tumour outgrowth without affecting wound healing processes. These new therapeutic approaches may be of importance especially after surgery or other invasive (diagnostic) procedures in cancer patients.

In vitro up-regulation of E-selectin and induction of interleukin-6 in endothelial cells by autoantibodies in Wegener's granulomatosis and microscopic polyangiitis.

OBJECTIVE: In patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA) autoantibodies to myeloid granule proteins (ANCA), particularly proteinase 3 (Pr3) and myeloperoxidase (MPO), and to endothelial cells (AECA) are frequently detected. The role of these autoantibodies in the development of vascular injury is incompletely understood. Since the expression of E-selectin and the production of interleukin 6 by endothelial cells is an early step in the sequence of events leading to vascular injury, we examined the capacity of IgG fractions from patients with WG and/or MPA to activate endothelial cells to the expression of E-selectin and the production of IL-6. We related those findings to the presence of ANCA and AECA in the IgG preparations. METHODS: Human umbilical vein endothelial cells (HUVEC) were incubated with immunoglobulin (IgG) preparations from 28 patients (17 positive for anti-Pr3, 10 for anti-MPO, and one for anti-Pr3/MPO) with active vasculitis and from 10 healthy volunteers. The final IgG concentration in the activation assay was 2 mg/ml. TNF alpha (10 ng/ml) and LPS (10 ng/ml) were used as positive controls for HUVEC activation. The extent of HUVEC activation was assessed by the measurement of E-selectin expression by flow cytometry (after 4 hours of incubation) and the production of interleukin 6 by ELISA (after 24 hours). RESULTS: We found that 11 of the 28 ANCA positive IgG samples were capable of activating endothelial cells: six samples induced IL-6 production alone, one sample upregulated E-selectin expression alone, and four samples induced both IL-6 production and E-selectin upregulation. Five of 17 anti-Pr3 positive samples (one of which was also positive for AECA) and 6 of 10 anti-MPO positive samples (all simultaneously positive for AECA) induced endothelial cell activation. AECA positive samples that induced endothelial cell activation (n = 7) had higher AECA titres than samples that did not induce endothelial cell activation (n = 6). CONCLUSION: Our data suggest that the activation of endothelial cells in patients with WG and MPA can be induced by circulating autoantibodies. Both ANCA and AECA can be responsible for this effect.

An SV40 large T-antigen immortalized human umbilical vein endothelial cell line for anti-endothelial cell antibody detection.

OBJECTIVE: Anti-endothelial cell antibodies in serum of patients with different inflammatory diseases can be detected by a whole cell enzyme-linked immunosorbant assay, using primary cultures of human umbilical vein endothelial cells. To avoid repeated isolation, it would be of great value if an immortal endothelial cell line could be used to perform anti-endothelial cell antibody assays. METHODS: In this study endothelial cells from human umbilical and iliac veins and arteries were transfected with a plasmid containing the Simian Virus 40 large T-antigen. Endothelial cell line(s) derived from this procedure were compared with human umbilical vein endothelial cells in the anti-endothelial cell antibody assay. RESULTS: After transfection, clones of homologous cell populations showed an extended lifespan, before entering a period of crisis. In one human umbilical vein endothelial cell clone a subpopulation of cells escaped crisis and became immortal (EVLC2). Telomerase was activated in this endothelial cell line, resulting in maintenance of the telomere length. There was a significant correlation between anti-endothelial cell antibody testing on human umbilical vein endothelial cells and on the cell line EVLC2. CONCLUSION: The Simian Virus 40 large T-antigen immortalized human umbilical vein endothelial cell line EVLC2 may be useful for the detection of anti-endothelial cell antibodies.

Targeting of naproxen covalently linked to HSA to sinusoidal cell types of the liver.

The kinetic behaviour of a naproxen human serum albumin conjugate (Nap23-HSA) was investigated in rats and in isolated perfused rat livers (IPRL), as compared to its active metabolite naproxen-lysine (Nap-lysine) and free naproxen. Through covalently linking the anti-inflammatory drug naproxen to HSA, this drug can be selectively delivered to non parenchymal cells of the liver. Liver endothelial and Kupffer cells play an important role in the pathogenesis of inflammatory liver diseases. Targeting naproxen to these cells might increase its efficacy and reduce the side effects. The altered kinetic properties of Nap23-HSA, after i.v. injection of 22 mg x kg(-1), as compared to an equimolar amount of the uncoupled drug, were demonstrated in vivo by a decrease in the steady state volume of distribution (41 +/- 5 vs. 134 +/- 19 ml x kg(-1)), a decrease in its clearance (0.48 +/- 0.05 vs. 0.63 +/- 0.1 ml x min(-1) x kg(-1)), a shorter plasma half life (60 +/- 11 vs. 152 +/- 44 min) and a sustained biliary excretion. Liver targeting of Nap23-HSA was clearly demonstrated: drug content of the liver 180 min after injection was about 30 times higher for Nap23-HSA as compared to naproxen itself. The IPRL experiments showed that the Vmax of hepatic removal of the conjugate was 40 microg x min(-1) x g liver(-1). With doses below receptor saturation a rapid removal of the conjugate (t1/2 = 6 min) from the perfusion medium was found. In conclusion, this study demonstrates the saturable uptake of Nap23-HSA and its lysosomal degradation in both in vivo and IPRL experiments. Covalently linked naproxen is released as Nap-lysine. This active metabolite accumulates in Kupffer and endothelial cells in which it reaches therapeutic concentrations. Release from these cells leads to rapid uptake by hepatocytes and carrier mediated excretion into bile. Levels of Nap-lysine in bile and plasma reflect the slowest step in its generation: the proteolytic release in endothelial and Kupffer cells.