A suite of genome-engineered hepatic cells provide novel insights into the spatiotemporal metabolism of APOB and APOB-containing lipoprotein secretion.

AIMS: APOB-containing very-low-density lipoprotein (VLDL) production, secretion, and clearance by hepatocytes is a central determinant of hepatic and circulating lipid levels. Impairment of any of the aforementioned processes is associated with the development of multiple diseases. Despite the discovery of genes and processes that govern hepatic VLDL metabolism, our understanding of the different mechanistic steps involved is far from complete. An impediment to these studies is the lack of tractable hepatocyte-based systems to interrogate and follow APOB in cells, which the current study addresses. METHODS AND RESULTS: To facilitate the cellular study of VLDL metabolism, we generated human hepatic HepG2 and Huh-7 cell lines in which CRISPR/Cas9-based genome engineering was used to introduce the fluorescent protein mNeonGreen into the APOB gene locus. This results in the production of APOB100-mNeon that localizes predominantly to the endoplasmic reticulum (ER) and Golgi by immunofluorescence and electron microscopy imaging. The production and secretion of APOB100-mNeon can be quantitatively followed in medium over time, and results in production of lipoproteins that are taken up via the LDLR pathway. Importantly, the production and secretion of APOB-mNeon is sensitive to established pharmacological and physiological treatments, and to genetic modifiers known to influence VLDL production in humans. As a showcase, we used HepG2-APOBmNeon cells to interrogate ER-associated degradation (ERAD) of APOB. Using a dedicated sgRNA library targeting all established membrane-associated ER-resident E3 ubiquitin ligases led to identification of SYNV1 as the E3 responsible for degradation of poorly-lipidated APOB in HepG2 cells. CONCLUSIONS: In summary, the engineered cells reported here allow the study of hepatic VLDL assembly and secretion, and facilitate spatiotemporal interrogation induced by pharmacologic and genetic perturbations.

Assessment of medullary and extramedullary myelopoiesis in cardiovascular diseases.

Recruitment of innate immune cells and their accumulation in the arterial wall and infarcted myocardium has been recognized as a central feature of atherosclerosis and cardiac ischemic injury, respectively. In both, steady state and under pathological conditions, majority of these cells have a finite life span and are continuously replenished from haematopoietic stem/progenitor cell pool residing in the bone marrow and extramedullary sites. While having a crucial role in the cardiovascular disease development, proliferation and differentiation of innate immune cells within haematopoietic compartments is greatly affected by the ongoing cardiovascular pathology. In the current review, we summarize key cells, processes and tissue compartments that are involved in myelopoiesis under the steady state, during atherosclerosis development and in myocardial infarction.

S100A9 Links Inflammation and Repair in Myocardial Infarction.

RATIONALE: The alarmin S100A9 has been identified as a potential therapeutic target in myocardial infarction. Short-term S100A9 blockade during the inflammatory phase post-myocardial infarction inhibits systemic and cardiac inflammation and improves cardiac function long term. OBJECTIVE: To evaluate the impact of S100A9 blockade on postischemic cardiac repair. METHODS AND RESULTS: We assessed cardiac function, hematopoietic response, and myeloid phagocyte dynamics in WT (wild type) C57BL/6 mice with permanent coronary artery ligation, treated with the specific S100A9 blocker ABR-238901 for 7 or 21 days. In contrast to the beneficial effects of short-term therapy, extended S100A9 blockade led to progressive deterioration of cardiac function and left ventricle dilation. The treatment reduced the proliferation of Lin-Sca-1+c-Kit+ hematopoietic stem and progenitor cells in the bone marrow and the production of proreparatory CD150+CD48-CCR2+ hematopoietic stem cells. Monocyte trafficking from the spleen to the myocardium and subsequent phenotype switching to reparatory Ly6CloMerTKhi macrophages was also impaired, leading to inefficient efferocytosis, accumulation of apoptotic cardiomyocytes, and a larger myocardial scar. The transcription factor Nur77 (Nr4a1 [nuclear receptor subfamily 4 group A member 1]) mediates the transition from inflammatory Ly6Chi monocytes to reparatory Ly6Clo macrophages. S100A9 upregulated the levels and activity of Nur77 in monocytes and macrophages in vitro and in Ly6Chi/int monocytes in vivo, and S100A9 blockade antagonized these effects. Finally, the presence of reparatory macrophages in the myocardium was also impaired in S100A9-/- mice with permanent myocardial ischemia, leading to depressed cardiac function long term. CONCLUSIONS: We show that S100A9 plays an important role in both the inflammatory and the reparatory immune responses to myocardial infarction. Long-term S100A9 blockade negatively impacts cardiac recovery and counterbalances the beneficial effects of short-term therapy. These results define a therapeutic window targeting the inflammatory phase for optimal effects of S100A9 blockade as potential immunomodulatory treatment in acute myocardial infarction.

Inhibition of pro-inflammatory myeloid cell responses by short-term S100A9 blockade improves cardiac function after myocardial infarction.

AIMS: Neutrophils have both detrimental and beneficial effects in myocardial infarction (MI), but little is known about the underlying pathways. S100A8/A9 is a pro-inflammatory alarmin abundantly expressed in neutrophils that is rapidly released in the myocardium and circulation after myocardial ischaemia. We investigated the role of S100A8/A9 in the innate immune response to MI. METHODS AND RESULTS: In 524 patients with acute coronary syndrome (ACS), we found that high plasma S100A8/A9 at the time of the acute event was associated with lower left ventricular ejection fraction (EF) at 1-year and increased hospitalization for heart failure (HF) during follow-up. In wild-type C57BL/6 mice with MI induced by permanent coronary artery ligation, treatment with the S100A9 blocker ABR-238901 during the inflammatory phase of the immune response inhibited haematopoietic stem cell proliferation and myeloid cell egression from the bone marrow. The treatment reduced the numbers of neutrophils and monocytes/macrophages in the myocardium, promoted an anti-inflammatory environment, and significantly improved cardiac function compared with MI controls. To mimic the clinical scenario, we further confirmed the effects of the treatment in a mouse model of ischaemia/reperfusion. Compared with untreated mice, 3-day ABR-238901 treatment significantly improved left ventricular EF (48% vs. 35%, P = 0.002) and cardiac output (15.7 vs. 11.1 mL/min, P = 0.002) by Day 21 post-MI. CONCLUSION: Short-term S100A9 blockade inhibits inflammation and improves cardiac function in murine models of MI. As an excessive S100A8/A9 release is linked to incident HF, S100A9 blockade might represent a feasible strategy to improve prognosis in ACS patients.

The SwissFEL soft X-ray free-electron laser beamline: Athos.

The SwissFEL soft X-ray free-electron laser (FEL) beamline Athos will be ready for user operation in 2021. Its design includes a novel layout of alternating magnetic chicanes and short undulator segments. Together with the APPLE X architecture of undulators, the Athos branch can be operated in different modes producing FEL beams with unique characteristics ranging from attosecond pulse length to high-power modes. Further space has been reserved for upgrades including modulators and an external seeding laser for better timing control. All of these schemes rely on state-of-the-art technologies described in this overview. The optical transport line distributing the FEL beam to the experimental stations was designed with the whole range of beam parameters in mind. Currently two experimental stations, one for condensed matter and quantum materials research and a second one for atomic, molecular and optical physics, chemical sciences and ultrafast single-particle imaging, are being laid out such that they can profit from the unique soft X-ray pulses produced in the Athos branch in an optimal way.

The soluble receptor for advanced glycation end-products (sRAGE) has a dual phase-dependent association with residual cardiovascular risk after an acute coronary event.

BACKGROUND AND AIMS: The pro-inflammatory alarmin S100A12 (EN-RAGE) and the soluble form of its receptor, the receptor for advanced glycation endproducts (sRAGE), have diverging roles in cardiovascular disease. In experimental studies, S100A12 promoted atherosclerosis while sRAGE treatment was anti-atherogenic and reduced myocardial infarction size by scavenging RAGE ligands. Here, we aimed to explore the links between S100A12, sRAGE, and long-term prognosis after an acute coronary syndrome (ACS). METHODS: We measured S100A12 and sRAGE in 524 patients within 24 h after an ACS, and again 6 weeks later in a subgroup of 114 patients. This subgroup also completed a follow-up echocardiography after 1 year. The median follow-up time for recurrent major adverse cardiovascular events (MACE), defined as recurrent ACS or cardiovascular death, was 25.7 ±â€¯12.6 months. RESULTS: In Cox proportional hazard analyses, baseline S100A12 and sRAGE were positively associated with the risk of MACE, independently of traditional cardiovascular risk factors. The association between sRAGE and MACE remained significant after additional adjustment for troponin T, NT-proBNP and hsCRP [HR 95%CI for highest versus lowest tertile 3.2 (1.5-6.5), p = 0.002]. High sRAGE was also associated with deteriorating left ventricular function and an increased rate of heart failure hospitalization post-discharge. In contrast, patients with increasing sRAGE at 6 weeks compared to baseline had lower incidence of recurrent ACS. CONCLUSIONS: Our data suggest that sRAGE has a dual, phase-dependent association with residual cardiovascular risk after ACS. These findings are important for the design and interpretation of future studies on sRAGE as biomarker and potential treatment in ACS patients.

High Plasma sRAGE (Soluble Receptor for Advanced Glycation End Products) Is Associated With Slower Carotid Intima-Media Thickness Progression and Lower Risk for First-Time Coronary Events and Mortality.

Objective- RAGE (receptor for advanced glycation end products) and EMMPRIN (extracellular matrix metalloproteinase inducer) are immune receptors for proinflammatory mediators. These receptors can also be found in a soluble form in the circulation. Soluble RAGE (sRAGE) has shown atheroprotective properties in animal studies, possibly by acting as a decoy receptor for its ligands. Whether sEMMPRIN (soluble EMMPRIN) has similar roles is unknown. We hypothesized that sRAGE and sEMMPRIN might be associated with vascular disease progression, incident coronary events, and mortality. Approach and Results- We measured baseline sRAGE and sEMMPRIN in 4612 cardiovascular disease-free individuals from the population-based Malmö Diet and Cancer cohort. Measurements of intima-media thickness in the common carotid artery were performed at inclusion and after a median of 16.5 years. sRAGE was negatively correlated with carotid intima-media thickness progression, independently of traditional cardiovascular risk factors, kidney function, and hsCRP (high sensitive C-reactive protein). Additionally, sRAGE was associated with decreased risk for major adverse coronary events (hazard ratio=0.90 [0.82-0.97]; P=0.009) and mortality (hazard ratio=0.93 [0.88-0.99]; P=0.011) during a follow-up period of 21 years. The relationship with mortality was independent of all considered potential confounders. We found no correlations between EMMPRIN, intima-media thickness progression, or prognosis. Conclusions- Individuals with high levels of circulating sRAGE have a slower rate of carotid artery disease progression and a better prognosis. Although its predictive value was too weak to promote sRAGE as a useful clinical biomarker in the population, the findings support further research into the potential anti-inflammatory and atheroprotective properties of this soluble receptor.

IL-1R and MyD88 signalling in CD4+ T cells promote Th17 immunity and atherosclerosis.

Aims: The role of CD4+ T cells in atherosclerosis has been shown to be dependent on cytokine cues that regulate lineage commitment into mature T helper sub-sets. In this study, we tested the roles of IL-1R1 and MyD88 signalling in CD4+ T cells in atherosclerosis. Methods and results: We transferred apoe-/-myd88+/+ or apoe-/-myd88-/- CD4+ T cells to T- and B-cell-deficient rag1-/-apoe-/- mice fed high fat diet. Mice given apoe-/-myd88-/- CD4+ T cells exhibited reduced atherosclerosis compared with mice given apoe-/-myd88+/+ CD4+ T cells. CD4+ T cells from apoe-/-myd88-/- produced less IL-17 but similar levels of IFN-γ. Treatment of human CD4+ T cells with a MyD88 inhibitor inhibited IL-17 secretion in vitro. Transfer of il1r1-/- CD4+ T cells recapitulated the phenotype seen by transfer of myd88-/- CD4+ T cells with reduced lesion development and a reduction in Th17 and IL-17 production compared with wild type CD4+ T cell recipients. Relative collagen content of lesions was reduced in mice receiving il1r1-/- CD4+ T cells. Conclusion: We demonstrate that both IL1R and MyD88 signalling in CD4+ T cells promote Th17 immunity, plaque growth and may regulate plaque collagen levels.

Murine Monocytes: Origins, Subsets, Fates, and Functions.

Monocytes are short-lived mononuclear phagocytes that circulate in the bloodstream and comprise two main subpopulations that in the mouse are best defined by the Ly6C marker. Intravascular functions of "classical" Ly6C+ monocytes and their interactions with other lymphoid and myeloid leukocytes in the circulation remain poorly understood. Rather, these cells are known to efficiently extravasate into tissues. Indeed, Ly6C+ monocytes and their descendants have emerged as a third, highly plastic and dynamic cellular system that complements the two classical, tissue-resident mononuclear phagocyte compartments, i.e., macrophages and dendritic cells, on demand. Following recruitment to injured tissue, Ly6C+ monocytes respond to local cues and can critically contribute to the initiation and resolution of inflammatory reactions. The second main murine monocyte subset, Ly6C- cells, derive in steady state from Ly6C+ monocytes and remain in the vasculature, where the cells act as scavengers. Moreover, a major fraction of Ly6C- monocytes adheres to the capillary endothelium and patrols the vessel wall for surveillance. Given the central role of monocytes in homeostasis and pathology, in-depth study of this cellular compartment can be highly informative on the health state of the organism and provides an attractive target for therapeutic intervention.

Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance.

BACKGROUND: The nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to modulate the inflammatory response of macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice. RESULTS: In line with previous observations, SDF-1α (CXCL12) was among the most upregulated genes in Nur77-deficient BMM and we demonstrated that Nur77 binds directly to the SDF-1α promoter, resulting in inhibition of SDF-1α expression. The cytokine receptor CX3CR1 was strongly downregulated in Nur77-KO BMM, implying involvement of Nur77 in macrophage tolerance. Ingenuity pathway analyses (IPA) to identify canonical pathways regulation and gene set enrichment analyses (GSEA) revealed a potential role for Nur77 in extracellular matrix homeostasis. Nur77-deficiency increased the collagen content of macrophage extracellular matrix through enhanced expression of several collagen subtypes and diminished matrix metalloproteinase (MMP)-9 activity. IPA upstream regulator analyses discerned the small GTPase Rac1 as a novel regulator of Nur77-mediated gene expression. We identified an inhibitory feedback loop with increased Rac1 activity in Nur77-KO BMM, which may explain the augmented phagocytic activity of these cells. Finally, we predict multiple chronic inflammatory diseases to be influenced by macrophage Nur77 expression. GSEA and IPA associated Nur77 to osteoarthritis, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, and allergic airway inflammatory diseases. CONCLUSIONS: Altogether these data identify Nur77 as a modulator of macrophage function and an interesting target to treat chronic inflammatory disease.

Stents Eluting 6-Mercaptopurine Reduce Neointima Formation and Inflammation while Enhancing Strut Coverage in Rabbits.

BACKGROUND: The introduction of drug-eluting stents (DES) has dramatically reduced restenosis rates compared with bare metal stents, but in-stent thrombosis remains a safety concern, necessitating prolonged dual anti-platelet therapy. The drug 6-Mercaptopurine (6-MP) has been shown to have beneficial effects in a cell-specific fashion on smooth muscle cells (SMC), endothelial cells and macrophages. We generated and analyzed a novel bioresorbable polymer coated DES, releasing 6-MP into the vessel wall, to reduce restenosis by inhibiting SMC proliferation and decreasing inflammation, without negatively affecting endothelialization of the stent surface. METHODS: Stents spray-coated with a bioresorbable polymer containing 0, 30 or 300 µg 6-MP were implanted in the iliac arteries of 17 male New Zealand White rabbits. Animals were euthanized for stent harvest 1 week after implantation for evaluation of cellular stent coverage and after 4 weeks for morphometric analyses of the lesions. RESULTS: Four weeks after implantation, the high dose of 6-MP attenuated restenosis with 16% compared to controls. Reduced neointima formation could at least partly be explained by an almost 2-fold induction of the cell cycle inhibiting kinase p27Kip1. Additionally, inflammation score, the quantification of RAM11-positive cells in the vessel wall, was significantly reduced in the high dose group with 23% compared to the control group. Evaluation with scanning electron microscopy showed 6-MP did not inhibit strut coverage 1 week after implantation. CONCLUSION: We demonstrate that novel stents coated with a bioresorbable polymer coating eluting 6-MP inhibit restenosis and attenuate inflammation, while stimulating endothelial coverage. The 6-MP-eluting stents demonstrate that inhibition of restenosis without leaving uncovered metal is feasible, bringing stents without risk of late thrombosis one step closer to the patient.

Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages.

Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn's disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.

The Ins and Outs of Small GTPase Rac1 in the Vasculature.

The Rho family of small GTPases forms a 20-member family within the Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well known Rho family members are RhoA (Ras homolog gene family, member A), Cdc42 (cell division control protein 42), and Rac1 (Ras-related C3 botulinum toxin substrate 1), which affect intracellular signaling pathways that regulate a plethora of critical cellular functions, such as oxidative stress, cellular contacts, migration, and proliferation. In this review, we describe the current knowledge on the role of GTPase Rac1 in the vasculature. Whereas most recent reviews focus on the role of vascular Rac1 in endothelial cells, in the present review we also highlight the functional involvement of Rac1 in other vascular cells types, namely, smooth muscle cells present in the media and fibroblasts located in the adventitia of the vessel wall. Collectively, this overview shows that Rac1 activity is involved in various functions within one cell type at distinct locations within the cell, and that there are overlapping but also cell type-specific functions in the vasculature. Chronically enhanced Rac1 activity seems to contribute to vascular pathology; however, Rac1 is essential to vascular homeostasis, which makes Rac1 inhibition as a therapeutic option a delicate balancing act.

6-Mercaptopurine reduces macrophage activation and gut epithelium proliferation through inhibition of GTPase Rac1.

BACKGROUND: Inflammatory bowel disease is characterized by chronic intestinal inflammation. Azathioprine and its metabolite 6-mercaptopurine (6-MP) are effective immunosuppressive drugs that are widely used in patients with inflammatory bowel disease. However, established understanding of their immunosuppressive mechanism is limited. Azathioprine and 6-MP have been shown to affect small GTPase Rac1 in T cells and endothelial cells, whereas the effect on macrophages and gut epithelial cells is unknown. METHODS: Macrophages (RAW cells) and gut epithelial cells (Caco-2 cells) were activated by cytokines and the effect on Rac1 signaling was assessed in the presence or absence of 6-MP. RESULTS: Rac1 is activated in macrophages and epithelial cells, and treatment with 6-MP resulted in Rac1 inhibition. In macrophages, interferon-γ induced downstream signaling through c-Jun-N-terminal Kinase (JNK) resulting in inducible nitric oxide synthase (iNOS) expression. iNOS expression was reduced by 6-MP in a Rac1-dependent manner. In epithelial cells, 6-MP efficiently inhibited tumor necrosis factor-α-induced expression of the chemokines CCL2 and interleukin-8, although only interleukin-8 expression was inhibited in a Rac1-dependent manner. In addition, activation of the transcription factor STAT3 was suppressed in a Rac1-dependent fashion by 6-MP, resulting in reduced proliferation of the epithelial cells due to diminished cyclin D1 expression. CONCLUSIONS: These data demonstrate that 6-MP affects macrophages and gut epithelial cells beneficially, in addition to T cells and endothelial cells. Furthermore, mechanistic insight is provided to support development of Rac1-specific inhibitors for clinical use in inflammatory bowel disease.

The LIM-only protein FHL2 reduces vascular lesion formation involving inhibition of proliferation and migration of smooth muscle cells.

The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.

Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug.

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.

Immunosuppressive drug azathioprine reduces aneurysm progression through inhibition of Rac1 and c-Jun-terminal-N-kinase in endothelial cells.

OBJECTIVE: In aortic aneurysms the arterial vessel wall is dilated because of destruction of its integrity, which may lead to lethal vessel rupture. Chronic infiltration of inflammatory cells into the vessel wall is fundamental to aneurysm pathology. We aim to limit aneurysm growth by inhibition of inflammation and reducing endothelial cell (EC) activation with immunosuppressive drug azathioprine (Aza). APPROACH AND RESULTS: Aza and its metabolite 6-mercaptopurine have anti-inflammatory effects on leukocytes. We here demonstrate that treatment of ECs with 6-mercaptopurine inhibits cell activation as illustrated by reduced expression of interleukin-12, CCL5, CCL2, and vascular cell adhesion molecule-1 and inhibition of monocyte-EC adhesion. The underlying mechanism of 6-mercaptopurine involves suppression of GTPase Rac1 activation, resulting in reduced phosphorylation of c-Jun-terminal-N-kinase and c-Jun. Subsequently, the effect of Aza was investigated in aneurysm formation in the angiotensin II aneurysm mouse model in apolipoprotein E-deficient mice. We demonstrated that Aza decreases de novo aortic aneurysm formation from an average aneurysm severity score of 2.1 (control group) to 0.6 (Aza group), and that Aza effectively delays aorta pathology in a progression experiment, resulting in a reduced severity score from 2.8 to 1.7 in Aza-treated mice. In line with the in vitro observations, Aza-treated mice showed less c-Jun-terminal-N-kinase activation in ECs and reduced leukocyte influx in the aortic wall. CONCLUSIONS: The immunosuppressive drug Aza has an anti-inflammatory effect and in ECs inhibits Rac1 and c-Jun-terminal-N-kinase activation, which may explain the protective effect of Aza in aneurysm development and, most importantly for clinical implications, aneurysm severity.

Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis.

RATIONALE: Nuclear receptor Nur77, also known as NR4A1, TR3, or NGFI-B, is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. Macrophages play a critical role in atherosclerosis and the function of Nur77 in lesion macrophages has not yet been investigated. OBJECTIVE: This study aims to delineate the function of Nur77 in macrophages and to assess the effect of bone marrow-specific deficiency of Nur77 on atherosclerosis. METHODS AND RESULTS: We investigated Nur77 in macrophage polarization using bone marrow-derived macrophages (BMM) from wild-type and Nur77-knockout (Nur77(-/-)) mice. Nur77(-/-) BMM exhibit changed expression of M2-specific markers and an inflammatory M1-phenotype with enhanced expression of interleukin-12, IFNγ, and SDF-1α and increased NO synthesis in (non)-stimulated Nur77(-/-) BMM cells. SDF-1α expression in nonstimulated Nur77(-/-) BMM is repressed by Nur77 and the chemoattractive activity of Nur77(-/-) BMM is abolished by SDF-1α inhibiting antibodies. Furthermore, Nur77(-/-) mice show enhanced thioglycollate-elicited migration of macrophages and B cells. The effect of bone marrow-specific deficiency of Nur77 on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Ldlr(-/-) mice with a Nur77(-/-)-deficient bone marrow transplant developed 2.1-fold larger atherosclerotic lesions than wild-type bone marrow-transplanted mice. These lesions contain more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF-1α expression is higher in lesions of Nur77(-/-)-transplanted mice, which may explain the observed aggravation of lesion formation. CONCLUSIONS: In conclusion, in bone marrow-derived cells the nuclear receptor Nur77 has an anti-inflammatory function, represses SDF-1α expression and inhibits atherosclerosis.