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The C1q/TNF-related protein 3 (CTRP3) represents a pleiotropic adipokine reciprocally associated with obesity and type 2 diabetes mellitus and exhibits anti-inflammatory properties in relation to lipopolysaccharides (LPS)-mediated effects in adipocytes, as well as monocytes/macrophages. Here, we focused on the influence of CTRP3 on LPS-mediated effects in endothelial cells in order to expand the understanding of a possible anti-inflammatory function of CTRP3 in a setting of endotoxemia. An organ- and tissue-specific expression analysis by real-time PCR revealed a considerable Ctrp3 expression in various adipose tissue compartments, however, higher levels were detected in the aorta and in abundantly perfused tissues (bone marrow and the thyroid gland). We observed a robust Ctrp3 expression in primary endothelial cells and a transient upregulation in murine endothelial (MyEND) cells by LPS (50 ng/mL). In MyEND cells, CTRP3 inhibited the LPS-induced expression of interleukin (Il)-6 and the tumor necrosis factor (Tnf)-α, and suppressed the LPS-dependent expression of the major endothelial adhesion molecules Vcam-1 and Icam-1. The LPS-induced adhesion of monocytic cells to an endothelial monolayer was antagonized by CTRP3. In C57BL/6J mice with an LPS-induced systemic inflammation, exogenous CTRP3 did not affect circulating levels of TNF-α, ICAM-1, and VCAM-1. In conclusion, we characterized CTRP3 beyond its function as an adipokine in a setting of vascular inflammation. CTRP3 inhibited LPS-induced endothelial expression of adhesion molecules and monocyte cell adhesion, indicating an important vascular anti-inflammatory role for CTRP3 in endotoxemia.

Im Rahmen dieser Arbeit wurden zwei separate Projekte bearbeitet: zum einen die NOD-abhängige miRNA Regulation und deren Einfluss auf die endotheliale Zytokinexpression als wesentlicher Bestandteil der vaskulären Inflammation. Zum anderen wurde die Rolle der Immunrezeptoren NOD1 und NOD2 innerhalb der Atherogenese und des Cholesterolmetabolismus charakterisiert. NOD1 und NOD2 gehören zur Proteinfamilie der Pattern Recognition Rezeptoren, welche für die Pathogenerkennung und nachgeschaltete angeborene Immunantwort relevant sind. Innerhalb des ersten Projektes wurden in murinen primären Lungenendothelzellen nach NOD Aktivierung drei differentiell regulierte miRNAs nachgewiesen: miR-147-3p, miR-200a-3p und miR-298-5p. Basierend auf der aktuellen Literatur und in dieser Arbeit durchgeführten in-silico Analysen konnten die Zytokine TNF-α und IL-6 als potentielle Zielgene der miRNAs miR-147-3p und miR-298-5p identifiziert werden. Dies wurde anhand einer verminderten Expression von TNF-α und IL-6 nach Transfektion mit entsprechenden miRNA mimics validiert. Da eine NOD Stimulation zur reduzierten Expression dieser beiden miRNAs führt, implizieren diese Ergebnisse einen miRNA-abhängigen, verstärkenden Effekt der NOD Stimulation auf endotheliale Zytokinexpression und vaskuläre Inflammation. Im zweiten Projekt konnte gezeigt werden, dass eine systemische Deletion von NOD1 und NOD2 zu verringerter Lipideinlagerung und Ansammlung von Monozyten/Makrophagen im atherosklerotischen Plaque führt. Dies geht mit reduzierten Plasma Cholesterolwerten einher und kann verschiedene Ursachen haben: zum einen zeigen Nod-defiziente Tiere eine erhöhte LRP1 Expression in der Leber, was auf eine verstärkte hepatische Cholesterolaufnahme hinweist. Makrophagen Nod-defizienter Mäuse weisen eine verstärkte Expression der Cholesteroltransporter ABCA1 und ABCG1 und eine reduzierte Schaumzellbildung auf, was einen gesteigerten reversen Cholesteroltransport in Nod-defizienten Mäusen nahelegt. Weiterhin konnte deutlich mehr Cholesterol und ein erhöhtes Vorkommen des Cholesterol metabolisierenden Darmbakteriums Eubacterium coprostanoligenes in Coecen Nod-defizienter Mäuse nachgewiesen werden. Diese Ergebnisse identifizieren NOD1 und NOD2 als essentielle Regulatoren innerhalb der Inflammation, des Cholesterolmetabolismus und der Atherogenese., Within the scope of this work two separate projects were investigated : the contribution of NOD-dependent microRNA regulation in endothelial cytokine expression and vascular inflammation and the role of NOD1 and NOD2 in atherogenesis and cholesterol metabolism. Both NOD1 and NOD2 receptors belong to the class of pattern recognition receptors responsible for pathogen sensing, which mediate a proinflammatory innate immune response upon their activation. Regarding the first project it was shown that NOD1 and NOD2 activation differentially regulates three different microRNAs in murine lung primary endothelial cells: miR-147-3p, miR-200a-3p and miR-298-5p. On the background of the current literature and in-silico analysis performed during this work, TNF-α and IL-6 were identified as potential target genes of miR-147-3p and miR-298-5p. By performing transfection experiments using mimics for both microRNAs TNF-α as well as IL-6 exhibited a diminished expression, indicating a microRNA-driven target gene regulation. Since miR-147-3p and miR-298-5p are downregulated after NOD-stimulation, these results point to an amplifying mechanism of NOD-dependent cytokine induction, an essential prerequisite of vascular inflammation. Within the second project it was demonstrated that deficiency of NOD1 and NOD2 results in reduced lipid and leukocyte accumulation in atherosclerotic plaques, which correlates with decreased plasma cholesterol. Potential causes of this Nod-deficient phenotype could be an enhanced hepatic cholesterol uptake, mediated by an increased expression of LRP1. Moreover, macrophages isolated from Nod-deficient mice showed reduced foam cell formation due to an increased ABCA1 and ABCG1 expression, thereby promoting reverse cholesterol transport. Finally, Nod-deficient mice showed increased cholesterol and coprostanol content in the cecum, which correlates with an elevated, even though not significant, relative abundance of the gut bacterium Eubacterium coprostanoligenes that metabolizes cholesterol to coprostanol. These results indicate a comprehensive interlinking role of NOD1 and NOD2, including microRNA-regulated vascular inflammation and metabolic processes affecting atherogenesis like cholesterol metabolism. These results identified NOD1 and NOD2 – at different levels – as comprehensive regulators in vascular inflammation and atherogenesis.

RATIONALE: Mechanistic insight into the inflammatory response after acute myocardial infarction (MI) may inform new molecularly-targeted treatment strategies to prevent chronic heart failure.; OBJECTIVE: We identified the sulfatase SULF2 in an in silico secretome analysis in bone marrow cells from patients with acute MI and detected increased sulfatase activity in myocardial autopsy samples. SULF2 (Sulf2 in mice) and its isoform SULF1 (Sulf1) act as endo-sulfatases removing 6 O sulfate groups from heparan sulfate (HS) in the extracellular space, thus eliminating docking sites for HS binding proteins. We hypothesized that the Sulfs have a role in tissue repair after MI.; METHODS AND RESULTS: Both Sulfs were dynamically upregulated after coronary artery ligation in mice, attaining peak expression and activity levels during the first week after injury. Sulf2 was expressed by monocytes and macrophages, Sulf1 by endothelial cells and fibroblasts. Infarct border-zone capillarization was impaired, scar size increased, and cardiac dysfunction more pronounced in mice with a genetic deletion of either Sulf1 or Sulf2. Studies in bone marrow-chimeric Sulf-deficient mice and Sulf-deficient cardiac endothelial cells established that inflammatory cell-derived Sulf2 and endothelial cell-autonomous Sulf1 promote angiogenesis. Mechanistically, both Sulfs reduced HS sulfation in the infarcted myocardium, thereby diminishing vascular endothelial growth factor A (Vegfa) interaction with HS. Along this line, both Sulfs rendered infarcted mouse heart explants responsive to the angiogenic effects of HS-binding Vegfa164 but did not modulate the angiogenic effects of non-HS-binding Vegfa120. Treating wild-type mice systemically with the small molecule HS antagonist surfen (bis 2 methyl-4-amino-quinolyl-6-carbamide, 1 mg/kg/day) for 7 days after MI released Vegfa from HS, enhanced infarct border-zone capillarization, and exerted sustained beneficial effects on cardiac function and survival.; CONCLUSIONS: These findings establish HS-editing Sulfs as critical inducers of postinfarction angiogenesis and identify HS sulfation as a therapeutic target for ischemic tissue repair.

Aim: NOD1 and 2 are intracellular pattern recognition receptors mediating vascular inflammatory processes. Since endothelial cells are crucially involved in vascular inflammation, we here investigated the microRNA (miRNA) expression profile in primary murine endothelial cells upon NOD stimulation and their role in endothelial cytokine expression. Methods: Primary lung endothelial cells were isolated from C57BL/6 mice (CD31/CD144), expanded and subjected to purity control (flow cytometry). Cells were stimulated with Tri-DAP (NOD1) or MDP (NOD2) respectively for 6h followed by RNA isolation. After RNA quality control, miRNA expression profiling was assessed by TaqMan Low Density Array. Raw data were analyzed using R Package HTqPCR (Bioconductor). Significantly regulated microRNAs were validated by qPCR and in-silico target gene analysis was performed (DIANA/miRPath, TargetScan, miRDB, miRTarBase). Target genes were analyzed performing miRNA transfection experiments and luciferase assays.Results: Array analysis revealed 10 NOD-dependently regulated miRNAs (Pu00b11.5). Two candidates, miR-147-3p and miR-298-5p, were validated by qPCR (P