Jochen Grommes

MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages

Macrophages in atherosclerotic plaques drive inflammatory responses, degrade lipoproteins, and phagocytose dead cells. MicroRNAs (miRs) control the differentiation and activity of macrophages by regulating the signaling of key transcription factors. However, the functional role of macrophage-related miRs in the immune response during atherogenesis is unknown. Here, we report that miR-155 is specifically expressed in atherosclerotic plaques and proinflammatory macrophages, where it was induced by treatment with mildly oxidized LDL (moxLDL) and IFN-γ. Leukocyte-specific Mir155 deficiency reduced plaque size and number of lesional macrophages after partial carotid ligation in atherosclerotic (Apoe-/-) mice. In macrophages stimulated with moxLDL/IFN-γ in vitro, and in lesional macrophages, loss of Mir155 reduced the expression of the chemokine CCL2, which promotes the recruitment of monocytes to atherosclerotic plaques. Additionally, we found that miR-155 directly repressed expression of BCL6, a transcription factor that attenuates proinflammatory NF-κB signaling. Silencing of Bcl6 in mice harboring Mir155-/- macrophages enhanced plaque formation and CCL2 expression. Taken together, these data demonstrated that miR-155 plays a key role in atherogenic programming of macrophages to sustain and enhance vascular inflammation.

MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1

Atherosclerosis, a hyperlipidemia-induced chronic inflammatory process of the arterial wall, develops preferentially at sites where disturbed laminar flow compromises endothelial cell (EC) function. Here we show that endothelial miR-126-5p maintains a proliferative reserve in ECs through suppression of the Notch1 inhibitor delta-like 1 homolog (Dlk1) and thereby prevents atherosclerotic lesion formation. Endothelial recovery after denudation was impaired in Mir126−/− mice because lack of miR-126-5p, but not miR-126-3p, reduced EC proliferation by derepressing Dlk1. At nonpredilection sites, high miR-126-5p levels in endothelial cells confer a proliferative reserve that compensates for the antiproliferative effects of hyperlipidemia, such that atherosclerosis was exacerbated in Mir126−/− mice. In contrast, downregulation of miR-126-5p by disturbed flow abrogated EC proliferation at predilection sites in response to hyperlipidemic stress through upregulation of Dlk1 expression. Administration of miR-126-5p rescued EC proliferation at predilection sites and limited atherosclerosis, introducing a potential therapeutic approach.

Disruption of Platelet-derived Chemokine Heteromers Prevents Neutrophil Extravasation in Acute Lung Injury

RATIONALE Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive. OBJECTIVES To determine mechanisms of platelet-mediated neutrophil recruitment in ALI. METHODS Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI. MEASUREMENTS AND MAIN RESULTS In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to P-selectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution. CONCLUSIONS Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference.

Presence of luminal neutrophil extracellular traps in atherosclerosis

Note: The editorial process for this article was fully handled by Prof. G. Y. H. Lip, Editor-in-Chief.

Simvastatin Reduces Endotoxin-Induced Acute Lung Injury by Decreasing Neutrophil Recruitment and Radical Formation

Introduction Treatment of acute lung injury (ALI) remains an unsolved problem in intensive care medicine. As simvastatin exerts protective effects in inflammatory diseases we explored its effects on development of ALI and due to the importance of neutrophils in ALI also on neutrophil effector functions. Methods C57Bl/6 mice were exposed to aerosolized LPS (500 µg/ml) for 30 min. The count of alveolar, interstitial, and intravasal neutrophils were assessed 4 h later by flow cytometry. Lung permeability changes were assessed by FITC-dextran clearance and albumin content in the BAL fluid. In vitro, we analyzed the effect of simvastatin on neutrophil adhesion, degranulation, apoptosis, and formation of reactive oxygen species. To monitor effects of simvastatin on bacterial clearance we performed phagocytosis and bacterial killing studies in vitro as well as sepsis experiments in mice. Results Simvastatin treatment before and after onset of ALI reduces neutrophil influx into the lung as well as lung permeability indicating the protective role of simvastatin in ALI. Moreover, simvastatin reduces the formation of ROS species and adhesion of neutrophils without affecting apoptosis, bacterial phagocytosis and bacterial clearance. Conclusion Simvastatin reduces recruitment and activation of neutrophils hereby protecting from LPS-induced ALI. Our results imply a potential role for statins in the management of ALI.

Annexin A1 Counteracts Chemokine-Induced Arterial Myeloid Cell Recruitment

RATIONALE Chemokine-controlled arterial leukocyte recruitment is a crucial process in atherosclerosis. Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor that recognizes proinflammatory and proresolving ligands. The contribution of FPR2 and its proresolving ligand annexin A1 to atherosclerotic lesion formation is largely undefined. OBJECTIVE Because of the ambivalence of FPR2 ligands, we here investigate the role of FPR2 and its resolving ligand annexin A1 in atherogenesis. METHODS AND RESULTS Deletion of FPR2 or its ligand annexin A1 enhances atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment. Mechanistically, we identify annexin A1 as an endogenous inhibitor of integrin activation evoked by the chemokines CCL5, CCL2, and CXCL1. Specifically, the annexin A1 fragment Ac2-26 counteracts conformational activation and clustering of integrins on myeloid cells evoked by CCL5, CCL2, and CXCL1 through inhibiting activation of the small GTPase Rap1. In vivo administration of Ac2-26 largely diminishes arterial recruitment of myeloid cells in a FPR2-dependent fashion. This effect is also observed in the presence of selective antagonists to CCR5, CCR2, or CXCR2, whereas Ac2-26 was without effect when all 3 chemokine receptors were antagonized simultaneously. Finally, repeated treatment with Ac2-26 reduces atherosclerotic lesion sizes and lesional macrophage accumulation. CONCLUSIONS Instructing the annexin A1-FPR2 axis harbors a novel approach to target arterial leukocyte recruitment. With the ability of Ac2-26 to counteract integrin activation exerted by various chemokines, delivery of Ac2-26 may be superior in inhibition of arterial leukocyte recruitment when compared with blocking individual chemokine receptors.

Safety and Feasibility of Ultrasound-accelerated Catheter-directed Thrombolysis in Deep Vein Thrombosis

OBJECTIVE One in four patients with primary iliofemoral deep vein thrombosis (DVT) develops post-thrombotic syndrome (PTS) within 1 year despite optimal standard anticoagulant therapy. Removal of thrombus by thrombolytic drugs may prevent PTS. The aim of this study was to assess the short-term safety and efficacy of ultrasound-accelerated catheter-directed thrombolysis (US-accelerated CDT). DESIGN This was a prospective non-randomised interventional study with US-accelerated CDT for DVT. PATIENTS AND METHODS Twelve patients with DVT (seven caval-iliofemoropopliteal, three iliofemoropopliteal, one femoropopliteal and one superior caval vein thrombosis) receiving standard anticoagulant and compression therapy, were treated with additional US-accelerated CDT (13 procedures) using the EKOS Endowave(®) system (EKOS Corporation, Bothell, WA, USA) between October 2008 and January 2010. RESULTS Thrombolysis was successful in 85% (11/13), with complete clot lysis (>90% restored patency) and in one case with partial clot lysis (50-90% restored patency). No pulmonary embolism and one bleeding at the catheter-insertion site were observed. In three patients, underlying lesions were successfully treated with balloon angioplasty and stent insertion. Four patients developed early recurrent thrombosis due to untreated residual venous obstruction. CONCLUSION US-accelerated CDT is a safe and promising treatment in patients with DVT. Residual venous obstruction should be treated by angioplasty and stent insertion to avoid early re-thrombosis.

Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4

MicroRNAs regulate the maladaptation of endothelial cells (ECs) to naturally occurring disturbed blood flow at arterial bifurcations resulting in arterial inflammation and atherosclerosis in response to hyperlipidemic stress. Here, we show that reduced endothelial expression of the RNAse Dicer, which generates almost all mature miRNAs, decreases monocyte adhesion, endothelial C–X–C motif chemokine 1 (CXCL1) expression, atherosclerosis and the lesional macrophage content in apolipoprotein E knockout mice (Apoe−/−) after exposure to a high-fat diet. Endothelial Dicer deficiency reduces the expression of unstable miRNAs, such as miR-103, and promotes Krüppel-like factor 4 (KLF4)-dependent gene expression in murine atherosclerotic arteries. MiR-103 mediated suppression of KLF4 increases monocyte adhesion to ECs by enhancing nuclear factor-κB-dependent CXCL1 expression. Inhibiting the interaction between miR-103 and KLF4 reduces atherosclerosis, lesional macrophage accumulation and endothelial CXCL1 expression. Overall, our study suggests that Dicer promotes endothelial maladaptation and atherosclerosis in part by miR-103-mediated suppression of KLF4.

Endothelial Hypoxia-Inducible Factor-1α Promotes Atherosclerosis and Monocyte Recruitment by Upregulating MicroRNA-19a

Chemokines mediate monocyte adhesion to dysfunctional endothelial cells (ECs) and promote arterial inflammation during atherosclerosis. Hypoxia-inducible factor (HIF)-1&agr; is expressed in various cell types of atherosclerotic lesions and is associated with lesional inflammation. However, the impact of endothelial HIF-1&agr; in atherosclerosis is unclear. HIF-1&agr; was detectable in the nucleus of ECs covering murine and human atherosclerotic lesions. To study the role of endothelial HIF-1&agr; in atherosclerosis, deletion of the Hif1a gene was induced in ECs from apolipoprotein E knockout mice (EC-Hif1a−/−) by Tamoxifen injection. The formation of atherosclerotic lesions, the lesional macrophage accumulation, and the expression of CXCL1 in ECs were reduced after partial carotid ligation in EC-Hif1a−/− compared with control mice. Moreover, the lesion area and the lesional macrophage accumulation were decreased in the aortas of EC-Hif1a−/− mice compared with control mice during diet-induced atherosclerosis. In vitro, mildly oxidized low-density lipoprotein or lysophosphatidic acid 20:4 increased endothelial CXCL1 expression and monocyte adhesion by inducing HIF-1&agr; expression. Moreover, endothelial Hif1a deficiency resulted in downregulation of miR-19a in atherosclerotic arteries determined by microRNA profiling. In vitro, HIF-1&agr;–induced miR-19a expression mediated the upregulation of CXCL1 in mildly oxidized low-density lipoprotein–stimulated ECs. These results indicate that hyperlipidemia upregulates HIF-1&agr; expression in ECs by mildly oxidized low-density lipoprotein–derived unsaturated lysophosphatidic acid. Endothelial HIF-1&agr; promoted atherosclerosis by triggering miR-19a–mediated CXCL1 expression and monocyte adhesion, indicating that inhibition of the endothelial HIF-1&agr;/miR-19a pathway may be a therapeutic option against atherosclerosis.

Minimally invasive treatment of chronic iliofemoral venous occlusive disease.

BACKGROUND As one of the primary etiologies of the post-thrombotic syndrome, chronic venous occlusion is a huge burden on patient quality of life and medical costs. In this study, we evaluate the short-term and midterm results of endovenous recanalization by angioplasty and stenting in chronic iliofemoral deep venous occlusions. METHODS This is a retrospective observational study set in a tertiary medical referral center. Patients with venous claudication or C4-6 venous disease combined with duplex and magnetic resonance-confirmed iliofemoral or caval occlusion were included. Patients with recent deep vein thrombosis (<1 year) were excluded. The intervention was endovascular deep venous recanalization, followed by angioplasty and stenting. Safety and feasibility were clinically evaluated during the procedure and during follow-up. Reocclusions and other treatment failures were evaluated during a maximum follow-up of 31 months by ultrasound imaging and venography. RESULTS Seventy-five procedures were performed in 63 patients (average age, 44 years; range, 18-75 years), of whom 86% had a history of deep venous thrombosis. The mean time between the initial deep venous thrombosis and treatment with PTA and stenting was 12 years (maximum, 31 years). May-Thurner syndrome was present in 57%. Forty-two procedures were performed in the left, six in the right, and 11 in both lower extremities. The vena cava inferior was partially stented in 25 patients. An average of 2.6 stents (median, 2) were used per procedure. Primary patency was 74% after 1 year. Assisted primary and secondary patency rates were 81% and 96%, respectively, at 1 year. Secondary procedures included restenting, catheter-directed thrombolysis, endophlebectomy of the common femoral vein, and creation of an arteriovenous fistula. No clinically evident pulmonary emboli were noted. A bleeding complication occurred after six procedures and was deemed major in two. No patients died. Relief or significant improvement of symptoms of chronic venous occlusive disease was achieved in 81% of patients. CONCLUSIONS Endovenous recanalization by angioplasty and stenting of chronically occluded iliofemoral vein segments is a safe and effective treatment with good short-term results, even when treatment takes place decades after the initial deep venous thrombosis. Most reocclusions can be adequately treated by a secondary procedure.