Dietger Stibenz

Furin-Like Proprotein Convertases Are Central Regulators of the Membrane Type Matrix Metalloproteinase–Pro-Matrix Metalloproteinase-2 Proteolytic Cascade in Atherosclerosis

Background—Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. Methods and Results—Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-&agr; or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP–dependent activation of vascular smooth muscle cell–derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. Conclusions—Furin-like PCs (furin and PC5) play a central role in a MT-MMP–MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

L-selectin is down-regulated in umbilical cord blood granulocytes and monocytes of newborn infants with acute bacterial infection.

ABSTRACT: The leukocyte glycoprotein l-selectin mediates an early step in the recruitment of leukocytes to sites of inflammation. l-Selectin surface expression is rapidly down-regulated by inflammatory signals in vitro. In a prospective study, we found l-selectin expression on umbilical cord blood granulocytes and monocytes to be significantly decreased in newborn infants with acute bacterial infection compared with controls (p < 0.01). A significantly reduced l-selectin expression of both granulocytes and monocytes was also found to be associated with an increased neutrophil immature/total ratio (p < 0.01) but not with other laboratory markers of neonatal sepsis. There was no apparent impact of prematurity, low birth weight, gestational hypertension, or gestational diabetes on l-selectin expression. Although the mode of delivery did not affect granulocyte l-selectin expression, umbilical cord blood monocytes showed an increased l-selectin expression after emergency cesarean delivery compared with samples obtained after elective cesarean or vaginal delivery (p < 0.01). We conclude that acute systemic inflammation results in down-regulation of granulocyte and monocyte l-selectin expression in vivo similar to that observed in vitro.

In Vivo Imaging of the Inflammatory Receptor CD40 After Cerebral Ischemia Using a Fluorescent Antibody

Background and Purpose— Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Methods— Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Results— Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. Conclusions— The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Soluble L-selectin (sCD62L) umbilical cord plasma levels increase with gestational age

ABSTRACT: L-Selectin (CD62L) is a leukocyte surface membrane glycoprotein involved in extravasation and homotypic aggregation which is rapidly cleaved off after cellular activation. From culture supernatants and body fluids, soluble L-selectin (sCD62L) has been recovered with its functional activity retained. We devised a sensitive enzyme-linked immunoassay for quantitation of sCD62L which was used to measure sCD62L in umbilical cord plasma of 255 human newborns with a gestational age (GA) of 23–43 wk (median 38 wk). sCD62L levels ranged from 1.14–13.8 pmol/mL (median 7.2 pmol/mL) and showed strong correlations with GA (r = 0.71, p < 0.001), birth weight (r = 0.66, p < 0.001), and absolute neutrophil cell counts (ANC) (r = 0.62, p < 0.001) obtained from a peripheral vein within the first 6 h of life (n—153), whereas there was a weak inverse correlation with absolute normoblast counts (r = —0.27, p < 0.001). In multiple regression analysis, only GA and ANC retained a significant association with sCD62L levels (p < 0.001). Decreased sCD62L levels were found to be associated with multiple gestation (4.8 ± 2.4 pmol/mL versus 7.7 ± 2.3 pmol/mL, p < 0.05) also when considering GA and ANC as covariates. In contrast, increased sCD62L levels in infants born from meconium-stained amniotic fluid, and decreased levels in newborns with acute bacterial infection could be fully attributed to differences in GA and ANC. Umbilical cord blood sCD62L levels of healthy, term, vaginally born singletons (n = 38) were significantly lower (8.5 ± 2.2 versus 11.8 ± 1.9, p < 0.0001) than cubital vein levels of healthy adults (n = 20). We conclude that sCD62L levels display a strong increase during fetal maturation.

CD45 Engagement Induces L‐Selectin Down‐Regulation

The CD45 glycoprotein isoforms exhibit a receptor‐like composition and display intracellular protein tyrosine phosphatase (PTPase) activity. The present study links CD45 to the regulation of L‐selectin (CD62L), a leucocyte glycoprotein important for extravasation and homotypic aggregation. Monoclonal antibodies (MoAbs) IOL1b and AICD45.2, but not GAP8.3, all of which are directed against common CD45 epitopes, were found to elicit lymphocyte L‐selectin down‐regulation. Lymphocyte L‐selectin down‐regulation in response to anti‐CD45 MoAbs was enhanced by high cell density and partially antagonized by the protein tyrosine kinase (PTK) inhibitor, herbimycin A. The MoAbs IOL1b, AICD45.2 and GAP8.3 recognized granulocyte‐expressed CD45 but did not induce loss of L‐selectin expression of granulocytes. In contrast, the CD45 PTPase inhibitor, vanadate, induced L‐selectin down‐regulation both in lymphocytes and granulocytes. The PTPase activation by nitric oxide (NO) or the NO‐generating compound, sodium nitroprusside, did not affect L‐selectin surface expression. Increased concentrations of soluble L‐selectin were detected after anti‐CD45 or vanadate‐induced down‐regulation of L‐selectin surface expression. While activation of protein kinase C (PKC) by phorbol 12‐myristate 13‐acetate (PMA) induces rapid L‐selectin down‐regulation of L‐selectin surface expression in both lymphocytes and granulocytes, the PKC inhibitor, H 7, was also found to down‐regulate lymphocyte and granulocyte L‐selectin surface expression. The inhibitor H 7 synergized with vanadate in down‐regulating lymphocyte L‐selectin surface expression, but partially inhibited vanadate‐induced granulocyte L‐selectin down‐regulation. The results suggest that in a cell type‐specific fashion the PKC system and tyrosine phosphorylation and dephosphorylation cascades are involved in the regulation of L‐selectin surface expression.

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

BACKGROUND CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration. METHODS AND RESULTS Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2-2.6 dyne/cm(2)) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of L-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated. CONCLUSION CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.

Integrin cleavage facilitates cell surface-associated proteolysis required for vascular smooth muscle cell invasion.

Vascular smooth muscle cell (VSMC) invasion is a key element in atherogenesis and restenosis, requiring integrins for adhesion/de-adhesion as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Among the MMP family, pro-MMP-2 is unique in its activation, depending on the formation of a multiprotein complex with MT1-MMP/TIMP-2 at the cell surface, in which integrin alphavbeta3 participates. Integrin alphav and MT1-MMP are synthesized from precursors via furin-dependent cleavage of their pro-peptide. Furin is the prototypical proprotein convertase highly expressed in VSMCs and human atherosclerotic lesions. Its precise role in the tight network involving MMPs/integrins and their coordination and cooperation required for VSMC invasion is unknown. We demonstrate that furin-inhibition with decanoyl-RVKR-chloromethylketone inhibits VSMC invasion in a comparable degree to MMP inhibitors, which reduce the MT1-MMP-MMP-2 proteolytic cascade. Furin-inhibition did not prevent MT1-MMP/MMP-2 maturation. In contrast, it strongly reduced pro-alphav cleavage, but did not lessen its cell membrane expression. However, inhibition of pro-alphav processing via furin-inhibition strongly reduced pro-MMP-2 binding to the cell surface, thereby lessening its full maturation and diminishing the cell surface in situ proteolysis required for invasion. Thus, our data demonstrate a novel mechanism of furin-dependent alphav cleavage that enhances pro-MMP-2 binding and activation at the cell membrane in cooperation with MT1-MMP in primary VSMCs. Processing of alphav by furin contributes to the recruitment of enzymatic energy to the cell surface, thereby providing focalized proteolysis associated with VSMC invasion.

Immunohistochemical localization of subtilisin/kexin-like proprotein convertases in human atherosclerosis

Integrins are heterodimeric α/β receptors that link the cytoskeleton with the extracellular matrix, thereby regulating several cell functions important in atherosclerosis. In vitro, the subtilisin/kexin-like proprotein convertases (PCs), namely PC5 and furin, have been shown to be responsible for the endoproteolytic activation of the αv integrin subunit. Based on their cleavage activity, these PCs are potential targets in atherosclerosis. In the present study, we investigated the localization of furin and PC5 in different stages of human atherosclerosis. Immunohistochemical analysis of furin and PC5 revealed their presence in vascular smooth-muscle cells and endothelial cells in atherosclerotic and non-atherosclerotic lesions. However, in the more advanced lesions, furin and PC5 staining was significantly expressed in macrophages/foam cells. In vitro, THP-1 derived macrophages contained furin and PC5, and maturation of monocytes to macrophages was accompanied by enhanced αvβ3 cell-surface expression. Inhibition of furin/PC5 with the specific pharmacological furin-like PC-inhibitor dec-CMK inhibited αv endoproteolytic activation but did not abolish αvβ3 cell-surface expression. This indicates that furin/PC5 is required for αv endoproteolytic activation but not for αv routing and sorting to the cell surface. In conclusion, our study demonstrates that furin and PC5 are significantly expressed in mononuclear cells in advanced human atherosclerotic lesions, where they regulate αv endoproteolytic activation.

Binding of human serum amyloid P componentto L-selectin

Serum concentrations of soluble L‐selectin by far exceed those of other soluble adhesion molecules, and serum soluble L‐selectin concentrations are remarkably stable upon prolonged storage. We present evidence for Ca2+‐dependent binding interactions between human serum amyloid P (SAP), a proteolysis‐resistant pentraxin glycoprotein, and L‐selectin, as shown by surface plasmon resonance measurements, protein band shift assays in a native PAGE system, and after SDS‐PAGE and membrane transfer. Monoclonal antibodies to L‐selectin strongly reduced binding of biotinylated SAP to L‐selectin‐IgG chimeras immobilized on microtiter plates. As binding was reduced by prior glycopeptidase F treatment of L‐selectin but not of SAP, it appears to be based on SAP lectin domain interactions with N‐linked L‐selectin carbohydrates. In freshly prepared human lymphocytes, SAP incubation induced expression of a β2 integrin neoepitope associated with high‐affinity binding. This was partially blocked by pre‐incubation with Fab fragments of two anti‐L‐selectin antibodies. In flow chamber experiments, SAP inhibited the adherence of human neutrophils to activated endothelium under shear stress. Thus, SAP binds to human L‐selectin and affects L‐selectin‐dependent leukocyte‐endothelial interactions.

Soluble l‐selectin (sCD62L) in relapsed childhood acute lymphoblastic leukaemia

Summary. Soluble l‐selectin (sCD62L) plasma concentrations at diagnosis and outcome were investigated in 193 children at first relapse of acute lymphoblastic leukaemia (ALL) after treatment according to the Berlin–Frankfurt–Münster relapsed ALL multicentre trials, ALL‐REZ BFM 95 and 96. sCD62L was low (< fifth paediatric reference percentile) in 63 (33%) and high (> 95th percentile) in 36 (19%) children, and was independent of remission duration, sex, BCR–ABL fusion or extramedullary disease. High sCD62L was associated with circulating blasts and T‐cell phenotype. More initial adverse events occurred in children with high and low levels of sCD62L (23 out of 99) than in those with normal levels (9 out of 94, P = 0·018). Among 75 worst‐prognosis patients (risk groups S3/S4, isolated bone marrow relapse occurring less than 6 months after elective cessation of front‐line therapy, or T‐cell phenotype with bone marrow involvement), 27 had low sCD62L and decreased event‐free survival (EFS) probability (PEFS5 = 0·09 at 5 years) and duration (219 d) compared with normal sCD62L (29 out of 75, PEFS5 = 0·24, 640 d, P = 0·01). Low (44 out of 118), normal (72 out of 118), and high (19 out of 118) sCD62L non‐S3/S4 patients fared similarly (average PEFS5 = 0·45, 1369 d; P = 0·5). Low sCD62L may be a marker of malignant blasts replacing normal sCD62L‐producing haematopoietic cells. In children with first relapse of ALL and worst prognosis, plasma sCD62L may be useful for risk‐adapted stratification.