Publications

ABSTRACT To define the role of the plasminogen activators (PAs) urokinase PA (uPA) and tissue PA (tPA) as well as the uPA receptor (uPAR) in arteriogenesis, we investigated their impact in a rabbit and mouse model of adaptive collateral artery growth. Collateral artery growth was induced by occlusion of the femoral artery in rabbit and wild‐type (WT) mice and in mice with targeted inactivation of uPA (uPA −/− ), tPA (tPA −/− ), or uPAR (uPAR −/− ). Northern blot results revealed a significant up‐regulation of uPA but not uPAR or tPA in the early phase of arteriogenesis in rabbit and WT mice. This up‐regulation on RNA level was followed by an increased protein level and enzymatic activity. Impaired perfusion recovery upon femoral artery ligation was observed by laser Doppler analysis in vivo in uPA‐deficient mice but not in uPAR or tPA deficiency compared with WT mice. Immunohistochemical studies revealed an association of leukocyte infiltration with arteriogenesis in WT mice that was strongly reduced in uPA −/− but not in uPAR‐ or tPA‐deficient mice. We conclude that arteriogenesis is promoted by an uPA‐mediated infiltration of leukocytes that is not dependent on uPAR.

Abstract Background Excessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia. Methods The dynamic of ROS levels was analysed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia ( ClinicalTrials.gov registration no. NCT02629406 ). The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes. Results Exposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD) - dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1) and was followed by functional consequences. The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes. Conclusions We conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are highly significant and timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use.

Cardiac hypertrophy, accompanied by progressive inflammation, and consequent heart failure (HF) continue to burden Western society. We show that miR‐155, a microRNA expressed by cardiac macrophages, stimulates the secretion of pro‐hypertrophic and ‐ inflammatory factors by macrophages and thereby causes adverse inflammation and HF. We subjected miR‐155 knockout (KO), wild type (WT) and antagomiR‐treated mice to pressure overload by angiotensin II or transverse aortic constriction to induce cardiac hypertrophy, inflammation and failure. Absence of miR‐155 inhibited the cardiac hypertrophic response and hampered cardiac macrophage activity. Bone marrow transplantation with miR‐155 KO and WT mice confirmed that miR‐155 in the macrophage mediates cardiac hypertrophy. It does so by repressing its direct target Suppressor of Cytokine Signalling‐1, leading to increased cytokine secretion and STAT3 activity, both implicated in cardiac inflammation and hypertrophy. These findings demonstrate the causative relation between inflammatory signalling and HF, and implicate microRNA‐155 as a therapeutic target in heart failure.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Abstract Late in erythropoiesis, nuclei are expelled from erythroblasts and engulfed by macrophages located in the blood island. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as a signal for their engulfment by macrophages. PS opsonins milk-fat-globule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors avb5 (and avb3) and Axl/Mertk/Tyro3, are involved in the phagocytosis of apoptotic cells. Because fetal liver and bone marrow macrophages do not express MFG-E8, GAS6-Mertk pathway might constitute a major pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from the spleens of phlebotomized mice (500 μl), and tested the capacity of bone marrow-derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), Axl (Axl−/−), Mertk (mertkkd) or Tyro3 (Tyro3−/−) to internalize these nuclei. Spleen erythroblasts were isolated 4 days after phlebotomy and cultured for 5 hours. Nuclei were obtained after disconnection from reticulocytes by mechanical shaking. Released nuclei were then identified by flow cytometry according to their size and their positive staining for the erythroid lineage marker Ter119 and Annexin V for PS labelling. Purity of the preparation was double-checked by morphological examination of cytospin preparations. Primary BMDM isolated from wild-type (WT) controls and GAS6−/−, Axl−/−, Mertkkd, Tyro3−/−, Axl/Tyro3−/−, Axl−/−/Mertkkd mice were incubated with nuclear preparation for 3 hours. BMDM were then washed to remove un-engulfed nuclei, analyzed in bright field and stained with May-Grünwald-Giemsa. Phagocytosis was determined by counting the number of BMDM with ingested nuclei and the phagocytosis index indicated the number of engulfed nuclei per macrophage. We found that GAS6−/− BMDM cleared 30% less nuclei than WT BMDM (p<0.01). We observed a slight decrease of internalization capacity for Axl−/− BMDM, whereas Tyro3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, Mertk deficiency nearly abolished the nuclei phagocytosis (p<0.001). Axl/Tyro3−/− and Axl−/−/Mertkkd BMDM were tested in comparison with WT BMDM and single knockouts, and did not show any cumulative effects when compared to single knockouts. Thus, Mertk was critical for the phagocytosis of nuclei from erythroblasts whereas the role of Axl and Tyro3 appeared to be negligible. In conclusion, we have shown that GAS6 and its receptor Mertk were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island. Indeed, GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and Mertk receptor on BMDM, allowing their efficient clearance.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.