Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

Aldosterone modulates endothelial permeability and endothelial nitric oxide synthase activity by rearrangement of the actin cytoskeleton.

Aldosterone (Aldo) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Aldo alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells were exposed to Aldo (10(-9) mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions and activation of intracellular signaling pathways were determined. Aldo increased endothelial permeability for molecules ≤ 70 kDa within 60 minutes. A transient loss of cortical actin with formation of actin stress fibers and disruption of continuous adherens and tight junction strands accompanied these changes. Mineralocorticoid receptor blockade, inhibition of RhoA, or disruption of extracellular-regulated protein kinase1/2 signaling pathways attenuated the Aldo-related effects. Moreover, Aldo-induced cytoskeletal rearrangement led to rapid dephosphorylation of protein kinase B and subsequent deactivation of endothelial nitric oxide synthase. Ex vivo tracer flux experiments with Evans blue-conjugated albumin demonstrated a concordant response to Aldo in freshly isolated umbilical arteries. Furthermore, low-dose cortisol (3 × 10(-10) to 3 × 10(-9) mol/L) mimics the effect of Aldo on endothelial integrity, and Aldo, by upregulating11ß-hydroxysteroid dehydrogenase type 2, might even aggravate this deleterious effect of low-dose cortisol. We suggest that these mechanisms may contribute to the vasculopathy induced by inappropriate mineralocorticoid receptor activation.

Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells.

BACKGROUND: Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC) which are widely used as model for endothelial cell-related studies. RESULTS: We here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP) and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na) and for longer incubation periods 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin. CONCLUSIONS: pCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins.

Endothelial-derived thrombospondin-1 promotes macrophage recruitment and apoptotic cell clearance.

Rapid apoptotic cell engulfment is crucial for prevention of inflammation and autoimmune diseases and is conducted by special immunocompetent cells like macrophages or immature dendritic cells. We recently demonstrated that endothelial cells (ECs) also participate in apoptotic cell clearance. However, in contrast to conventional phagocytes they respond with an inflammatory phenotype. To further confirm these pro-inflammatory responses human ECs were exposed to apoptotic murine ECs and changes in thrombospondin-1 (TSP-1) expression and in activation of intracellular signalling cascades were determined by real-time qPCR, immunoblotting and immunocytochemistry. Human primary macrophages or monocytic lymphoma cells (U937) were incubated with conditioned supernatant of human ECs exposed to apoptotic cells and changes in activation, migration and phagocytosis were monitored. Finally, plasma levels of TSP-1 in patients with anti-neutrophil cytoplasmic antibody(ANCA)-associated vasculitis (AAV) were determined by ELISA. We provided evidence that apoptotic cells induce enhanced expression of TSP-1 in human ECs and that this increase in TSP-1 is mediated by the mitogen-activated protein kinases (MAPK) ERK1 and 2 and their upstream regulators MEK and B-Raf. We also showed that plasma TSP-1 levels are increased in patients with AAV. Finally, we showed that conditioned supernatant of ECs exposed to apoptotic cells induces pro-inflammatory responses in monocytes or U937 cells and demonstrated that increased TSP-1 expression enhances migration and facilitates engulfment of apoptotic cells by monocyte-derived macrophages or U937 cells. These findings suggest that under pathological conditions with high numbers of uncleared dying cells in the circulation endothelial-derived elevated TSP-1 level may serve as an attraction signal for phagocytes promoting enhanced recognition and clearance of apoptotic cells.

Engulfment of apoptotic cells by microvascular endothelial cells induces proinflammatory responses.

Circulating endothelial cells (CECs) have been detected in a variety of vascular disorders, but their interactions with healthy endothelium remain unknown. The aim of this study was to evaluate the response of human endothelial cells (ECs) to apoptotic or necrotic ECs in an in vitro model and to delineate pathogenetic pathways. Here we show that incubation of the human microvascular endothelial cell line (HMEC-1) with apoptotic ECs resulted in increased expression of chemokines and enhanced binding of leukocytes to HMEC-1 cells, whereas exposure of HMEC-1 cells to necrotic ECs caused no changes in leukocyte-binding affinity. Both apoptotic and necrotic cells were bound and engulfed by HMEC-1 cells and primary human umbilical vein endothelial cells (HUVECs). We therefore suggest that exposures to apoptotic and necrotic ECs induce different patterns of chemokine synthesis and leukocyte adhesion in healthy ECs. These data indicate that CECs are not only markers of vascular damage but may induce proinflammatory signals in the endothelium.