MiR-21 Deficiency Alters the Survival of Ly-6Clo Monocytes in ApoE -/- Mice and Reduces Early-Stage Atherosclerosis.
Anna ChipontBruno EspositoInès ChallierMélanie MontabordAlain TedguiZiad MallatXavier LoyerStéphane Potteaux
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Apolipoprotein E
Chondroitin sulphate (CS) has long been used to treat osteoarthritis. Some investigations have also shown that the treatment with CS could reduce coronary events in patients with heart disease but no studies have identified the mechanistic role of these therapeutic effects. We aimed to investigate how the treatment with CS can interfere with the progress of atherosclerosis. The aortic arch, thoracic aorta and serum were obtained from apolipoprotein E (ApoE) knockout mice fed for 10 weeks with high-fat diet and then treated with CS (300 mg/kg, n = 15) or vehicle (n = 15) for 4 weeks. Atheromatous plaques were highlighted in aortas with Oil Red staining and analysed by microscopy. ApoE knockout mice treated with CS exhibited attenuated atheroma lesion size by 68% as compared with animals receiving vehicle. Serum lipids, glucose and C-reactive protein were not affected by treatment with CS. To investigate whether CS locally affects the inflamed endothelium or the formation of foam cells in plaques, human endothelial cells and monocytes were stimulated with tumour necrosis factor α or phorbol myristate acetate in the presence or absence of CS. CS reduced the expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 and ephrin-B2 and improved the migration of inflamed endothelial cells. CS inhibited foam cell formation in vivo and concomitantly CD36 and CD146 expression and oxidized low-density lipoprotein uptake and accumulation in cultured activated human monocytes and macrophages. Reported cardioprotective effects of CS may arise from modulation of pro-inflammatory activation of endothelium and monocytes and foam cell formation.
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Inflammatory responses are the driving force of atherosclerosis development. IκB kinase β (IKKβ), a central coordinator in inflammation through regulation of nuclear factor-κB, has been implicated in the pathogenesis of atherosclerosis. Macrophages play an essential role in the initiation and progression of atherosclerosis, yet the role of macrophage IKKβ in atherosclerosis remains elusive and controversial. This study aims to investigate the impact of IKKβ expression on macrophage functions and to assess the effect of myeloid-specific IKKβ deletion on atherosclerosis development.To explore the issue of macrophage IKKβ involvement of atherogenesis, we generated myeloid-specific IKKβ-deficient low-density lipoprotein receptor-deficient mice (IKKβ(ΔMye)LDLR(-/-)). Deficiency of IKKβ in myeloid cells did not affect plasma lipid levels but significantly decreased diet-induced atherosclerotic lesion areas in the aortic root, brachiocephalic artery, and aortic arch of low-density lipoprotein receptor-deficient mice. Ablation of myeloid IKKβ attenuated macrophage inflammatory responses and decreased atherosclerotic lesional inflammation. Furthermore, deficiency of IKKβ decreased adhesion, migration, and lipid uptake in macrophages.The present study demonstrates a pivotal role for myeloid IKKβ expression in atherosclerosis by modulating macrophage functions involved in atherogenesis. These results suggest that inhibiting nuclear factor-κB activation in macrophages may represent a feasible approach to combat atherosclerosis.
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New evidence demonstrates that aging and dyslipidemia are closely associated with oxidative stress, DNA damage and apoptosis in some cells and extravascular tissues. However, in monocytes, which are naturally involved in progression and/or resolution of plaque in atherosclerosis, this concurrence has not yet been fully investigated. In this study, we evaluated the influence of aging and hypercholesterolemia on serum pro-inflammatory cytokines, oxidative stress, DNA damage and apoptosis in monocytes from apolipoprotein E-deficient (apoE-/-) mice compared with age-matched wild-type C57BL/6 (WT) mice. Experiments were performed in young (2-months) and in old (18-months) male wild-type (WT) and apoE-/- mice.Besides the expected differences in serum lipid profile and plaque formation, we observed that atherosclerotic mice exhibited a significant increase in monocytosis and in serum levels of pro-inflammatory cytokines compared to WT mice. Moreover, it was observed that the overproduction of ROS, led to an increased DNA fragmentation and, consequently, apoptosis in monocytes from normocholesterolemic old mice, which was aggravated in age-matched atherosclerotic mice.In this study, we demonstrate that a pro-inflammatory systemic status is associated with an impairment of functionality of monocytes during aging and that these parameters are fundamental extra-arterial contributors to the aggravation of atherosclerosis. The present data open new avenues for the development of future strategies with the purpose of treating atherosclerosis.
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Aims: Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated LDL levels have been associated with activation of monocytes, intensive LDL lowering may reverse these pro-inflammatory changes. Subjects with elevated LDL levels are currently treated with statins, which are also described to have pleiotropic effects. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies which selectively reduce LDL we studied the impact of LDL lowering on monocyte phenotype and function in patients with familial hypercholesterolemia (FH). Methods and Results: We assessed monocyte phenotype and function using flow cytometry for a broad range of monocyte-relevant markers and a trans-endothelial migration assay in FH patients (n=22: LDL-C 6.8±1.9mmol/L) and healthy controls (n=18, LDL-C 2.9±0.8mmol/L). Interestingly, monocyte chemokine receptor (CCR) 2 expression was approximately 3-fold increased in FH patients compared with controls (ΔMFI 605±214 vs 236±155 P <0.001). CCR2 expression correlated significantly with plasma LDL-C levels (r=0.709) and positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity ex vivo. After 24 weeks of PCSK9 monoclonal antibody treatment (n=17), plasma LDL-C was reduced by 49% (from 6.7±1.3 mmol/L to 3.4±1.3 mmol/L P <0.001), which coincided with reduced monocyte intracellular lipid accumulation and suppressed CCR2 expression (ΔMFI: baseline 607±209, post PCSK9 mAbs: 207±180, P <0.001). Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 monoclonal antibody therapy. All changes were comparable in subjects who were treated with statins (n=14: LDL-C 2.8±0.6mmol/L) indicating that the anti-inflammatory effects were mainly mediated through LDL lowering. Conclusions: Elevated LDL levels in FH induce pro-inflammatory changes in monocytes, which is dampened by PCSK9 monoclonal antibody therapy. LDL lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL lowering itself is associated with anti-inflammatory effects on circulating monocytes.
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Abstract Aims Here, we aimed to determine the therapeutic effect of longevity-associated variant (LAV)-BPIFB4 gene therapy on atherosclerosis. Methods and results ApoE knockout mice (ApoE−/−) fed a high-fat diet were randomly allocated to receive LAV-BPIFB4, wild-type (WT)-BPIFB4, or empty vector via adeno-associated viral vector injection. The primary endpoints of the study were to assess (i) vascular reactivity and (ii) atherosclerotic disease severity, by Echo-Doppler imaging, histology and ultrastructural analysis. Moreover, we assessed the capacity of the LAV-BPIFB4 protein to shift monocyte-derived macrophages of atherosclerotic mice and patients towards an anti-inflammatory phenotype. LAV-BPIFB4 gene therapy rescued endothelial function of mesenteric and femoral arteries from ApoE−/− mice; this effect was blunted by AMD3100, a CXC chemokine receptor type 4 (CXCR4) inhibitor. LAV-BPIFB4-treated mice showed a CXCR4-mediated shift in the balance between Ly6Chigh/Ly6Clow monocytes and M2/M1 macrophages, along with decreased T cell proliferation and elevated circulating levels of interleukins IL-23 and IL-27. In vitro conditioning with LAV-BPIFB4 protein of macrophages from atherosclerotic patients resulted in a CXCR4-dependent M2 polarization phenotype. Furthermore, LAV-BPIFB4 treatment of arteries explanted from atherosclerotic patients increased the release of atheroprotective IL-33, while inhibiting the release of pro-inflammatory IL-1β, inducing endothelial nitric oxide synthase phosphorylation and restoring endothelial function. Finally, significantly lower plasma BPIFB4 was detected in patients with pathological carotid stenosis (>25%) and intima media thickness >2 mm. Conclusion Transfer of the LAV of BPIFB4 reduces the atherogenic process and skews macrophages towards an M2-resolving phenotype through modulation of CXCR4, thus opening up novel therapeutic possibilities in cardiovascular disease.
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Background The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses. Factors implicated in the development of atherosclerosis including modified low-density lipoprotein (LDL), cytokines and even shear stress induce p38 activation in endothelial cells and macrophages, which may be important for plaque formation. This study investigates the effects of endothelial- and macrophage-specific deficiency of p38α in atherosclerosis development, in Apolipoprotein E deficient (ApoE−/−) mice. Methodology/Principal Findings ApoE−/− mice with macrophage or endothelial cell-specific p38α deficiency were fed a high cholesterol diet (HCD) for 10 weeks and atherosclerosis development was assessed by histological and molecular methods. Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development. Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE−/− mice. Conclusions Although previous studies implicated p38α signaling in atherosclerosis, our in vivo experiments suggest that p38α function in endothelial cells and macrophages does not play an important role in atherosclerotic plaque formation in ApoE deficient mice.
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Transplant arteriosclerosis (TA) restricts long-term survival of heart transplant recipients. Although the role of monocyte/macrophages is well established in native atherosclerosis, it has been studied to a much lesser extent in TA. Plasma cholesterol is the most important non-immunologic risk factor for development of TA but the underlying mechanisms are largely unknown. We hypothesized that monocyte/macrophages might play an important role in the pathogenesis of TA under hyperlipidemic conditions.We studied TA in fully mismatched arterial allografts transplanted into hyperlipidemic ApoE(-/-) recipients compared to wild-type controls. The recruitment of distinct monocyte populations into the grafts was tracked by in vivo labelling with fluorescent microspheres. We used antibody-mediated depletion protocols to dissect the relative contribution of T lymphocytes and monocytes to disease development.In the hyperlipidemic environment the progression of TA was highly exacerbated and the inflammatory CD11b(+)CD115(+)Ly-6C(hi) monocytes were preferentially recruited into the neointima. The number of macrophage-derived foam cells present in the grafts strongly correlated with plasma cholesterol and disease severity. Depletion of Ly-6C(hi) monocytes and neutrophils significantly inhibited macrophage accumulation and disease progression. The accelerated monocyte recruitment occurs through a T cell-independent mechanism, as T cell depletion did not influence macrophage accumulation into the grafts.Our study identifies for the first time the involvement of inflammatory Ly-6C(hi) monocytes into the pathogenesis of TA, particularly in conditions of hyperlipidemia. Targeted therapies modulating the recruitment and activation of these cells could potentially delay coronary allograft vasculopathy and improve long-term survival of heart transplant recipients.
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Hyperlipidemia (HL) is a major cardiovascular risk factor promoting atherogenesis leading to coronary and peripheral artery disease. Collateral vessel development, a crucial compensatory mechanism in peripheral artery disease, is negatively influenced by HL [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Monocytes and tissue-resident macrophages accelerate both atherogenesis and arteriogenesis by supplying growth factors, cytokines and proteolytic enzymes [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. The decreased monocyte/macrophage accumulation around growing collateral vessels in HL is associated with jeopardized arteriogenesis [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar].Ligand-induced monocyte extravasation to sites of collateral growth is a crucial step. This involves several factors including monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial factor-A (VEGF-A), which facilitate monocyte chemotaxis in arteriogenesis, a short lasting and well defined process [2Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar, 3Tchaikovski V. Fellbrich G. Waltenberger J. The molecular basis of VEGFR-1 signal transduction pathways in primary human monocytes.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 322-328Crossref PubMed Scopus (87) Google Scholar]. Previous studies in humans indicated that cardiovascular risk factors such as HL severely hamper ex vivo monocyte chemotaxis to VEGF-A and MCP-1 [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar]. Based on these findings, we proposed that monocytes can be used as circulating "biosensors" to detect metabolic deregulation and elevated cardiovascular risk [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar]. However, HL often co-exists with other cardiovascular risk factors such as diabetes mellitus (DM) or smoking, all known to negatively affect monocyte chemotaxis [4Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar, 5Tchaikovski V. Olieslagers S. Bohmer F.D. Waltenberger J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes.Circulation. 2009; 120: 150-159Crossref PubMed Scopus (87) Google Scholar].We now demonstrate that monocytes from ApoE−/− mice with HL show a completely abrogated chemotaxis towards VEGF-A and a significantly decreased one towards MCP-1. Experiments were approved by the local animal ethical committee of Maastricht University. Blood was sampled [[6]Tchaikovski S.N. van Vlijmen B.J. Rosing J. Tans G. Development of a calibrated automated thrombography based thrombin generation test in mouse plasma.J. Thromb. Haemost. 2007; 5: 2079-2086Crossref PubMed Scopus (53) Google Scholar] and white blood cell counts were performed. Lipid values were measured enzymatically. Plasma lipid levels were significantly elevated in ApoE−/− mice (Table 1). ApoE−/− mice had significantly higher numbers of CD11b + monocytes (p < 0.05).Table 1Characteristics of mice, plasma lipid levels and monocyte numbers.WT (n = 34)ApoE−/− (n = 34)p-ValueAge of mice (weeks)38.9 ± 8.437.8 ± 10.1n.s.Total cholesterol (mg/dL)55.07 ± 7.97399.94 ± 85.8p < 0.05LDL cholesterol (mg/dL)5.92 ± 2.89278.46 ± 64.29p < 0.05HDL cholesterol (mg/dL)41.37 ± 6.5598.18 ± 16.24p < 0.05Triglicerides (mg/dL)46.05 ± 12.71116.53 ± 56.29p < 0.05Mononuclear cells (MNC, ×106/mL)1.47 ± 0.321.59 ± 0.47n.s.CD11b + monocytes (% of MNC)6.35 ± 0.8611.97 ± 2.26*p < 0.05LDL — low density lipoprotein, HDL — high density lipoprotein. Open table in a new tab For chemotaxis analysis blood from sex, age and genetically (C57Bl/6 or ApoE−/−) matched mice (avg. 5–6 mice per experimental condition, each condition was repeated 3–6 times — "n" in Fig. 1) was pooled and chemotaxis analysis of isolated monocytes was performed [[7]Kerber M. Reiss Y. Wickersheim A. Jugold M. Kiessling F. Heil M. et al.Flt-1 signaling in macrophages promotes glioma growth in vivo.Cancer Res. 2008; 68: 7342-7351Crossref PubMed Scopus (134) Google Scholar]. Monocytes from ApoE−/− mice show significantly impaired chemotactic responses to VEGF-A and MCP-1 compared to wild-type (WT) mice (Fig. 1A, B). In fact, chemotaxis to VEGF-A was not distinguishable from chemokinesis. In contrast, MCP-1-induced chemotaxis was reduced with only a moderate stimulation at the optimal concentration of 10 ng/mL (p < 0.05). Furthermore, monocytes from ApoE−/− mice had a significantly elevated chemokinesis (Fig. 1C) as compared to WT mice (p < 0.05).The pioneering finding of this study is that HL-conditioned monocytes from ApoE−/− mice are dysfunctional, as chemotaxis towards both VEGF-A or MCP-1 is severely impaired. These data imply that i.) monocytes show a functional defect in the presence of HL, and that ii.) monocyte dysfunction is likely to contribute to pathological changes observed in ApoE−/− mice [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar].Besides stimulation of atherogenesis [[8]Hansson G.K. Hermansson A. The immune system in atherosclerosis.Nat. Immunol. 2011; 12: 204-212Crossref PubMed Scopus (1546) Google Scholar], chronic HL impairs arteriogenesis [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Monocytes contribute to arteriogenesis by VEGFR-1- [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar] or CCR-2-mediated [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar] migration from the blood stream to the growing vessel [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. Reduced expression of arteriogenic factors is an unlikely cause for impaired arteriogenesis in HL as therapeutic rescue attempts with either VEGF-A or MCP-1 largely failed [1Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar, 9van Royen N. Hoefer I. Buschmann I. Kostin S. Voskuil M. Bode C. et al.Effects of local MCP-1 protein therapy on the development of the collateral circulation and atherosclerosis in Watanabe hyperlipidemic rabbits.Cardiovasc. Res. 2003; 57: 178-185Crossref PubMed Scopus (64) Google Scholar]. Furthermore, impaired arteriogenesis in HL mice was accompanied by decreased recruitment of macrophages both under the HL conditions and following the arteriogenic stimulation [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. This previous finding implies – in the light of our novel data – that both native as well as growth factor-stimulated arteriogenesis are reduced in HL due to a reduced/delayed accumulation of dysfunctional blood-derived monocytes at sites of vascular repair [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Indeed, bone marrow-derived cells from WT mice alleviate hindlimb ischemia in ApoE−/− mice by improving blood flow and promoting arteriogenesis [[10]Terry T. Chen Z. Dixon R.A. Vanderslice P. Zoldhelyi P. Willerson J.T. et al.CD34/M-cadherin bone marrow progenitor cells promote arteriogenesis in ischemic hindlimbs of ApoE mice.PLoS One. 2011; 6: e20673Crossref PubMed Scopus (14) Google Scholar].Our previous work on human monocytes documented an impaired chemotactic response to arteriogenic ligands VEGF-A and MCP-1 [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar] in HL. Our novel findings proof the same in ApoE−/− mice. Impaired monocyte chemotaxis to VEGF-A contributes to impaired arteriogenesis in DM [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar]. DM induces unspecific monocyte activation secondary to increased oxidative stress and advanced glycation of functionally relevant molecules [[5]Tchaikovski V. Olieslagers S. Bohmer F.D. Waltenberger J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes.Circulation. 2009; 120: 150-159Crossref PubMed Scopus (87) Google Scholar]. The described monocyte dysfunction in HL may be due to monocyte activation following lipid overload-induced oxidative stress and leads to up-regulation of adhesion molecules [[11]Tabas I. The role of endoplasmic reticulum stress in the progression of atherosclerosis.Circ. Res. 2010; 107: 839-850Crossref PubMed Scopus (364) Google Scholar]. This activation of monocytes may explain the observed elevated chemokinesis in ApoE−/− mice (Fig. 1C) and extravasation to inflamed endothelium of atherosclerotic plaques. Altogether, increased adhesive properties, increased monocyte numbers (Table 1) and extensive intraplaque angiogenesis will promote atherogenesis by progressive monocyte migration to the lesion [[8]Hansson G.K. Hermansson A. The immune system in atherosclerosis.Nat. Immunol. 2011; 12: 204-212Crossref PubMed Scopus (1546) Google Scholar] despite partially impaired migration to pro-atherogenic stimuli (here tested MCP-1). A broad spectrum of chemokines is responsible for monocyte recruitment in atherosclerosis. Modified lipoproteins may serve as chemoattractants [[8]Hansson G.K. Hermansson A. The immune system in atherosclerosis.Nat. Immunol. 2011; 12: 204-212Crossref PubMed Scopus (1546) Google Scholar] as well. Therefore, therapeutic strategies may need to target multiple targets.In the process of compensatory arteriogenesis the stimuli for monocyte recruitment and the therapeutic time window (days/weeks) are rather short compared to atherosclerosis (years/decades) [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. Therefore a decreased monocyte response demonstrated in our study may provide a functional basis for impaired/delayed arteriogenesis while atherogenesis continues.Lowering monocyte numbers can block the progression of atherosclerosis [[12]Swirski F.K. Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure.Science. 2013; 339: 161-166Crossref PubMed Scopus (699) Google Scholar]. The applicability, however, is limited by the fact that post-infarction monocytosis is physiologically required for both healing the damaged myocardium [[13]Dutta P. Courties G. Wei Y. Leuschner F. Gorbatov R. Robbins C.S. et al.Myocardial infarction accelerates atherosclerosis.Nature. 2012; 487: 325-329Crossref PubMed Scopus (727) Google Scholar] and for neovascularisation [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. These findings point towards the dilemma of why and how monocytes promote tissue healing in infarcted myocardium and in parallel worsen atherosclerosis. Monocytosis is an accompanying condition featuring several cardiovascular risk factors (e.g., HL, DM) in which the healing capability of monocytes is disturbed. Our findings stress the importance of therapeutically improving monocyte responsiveness to "healing" stimuli such as VEGF, which then could allow to therapeutically lower monocyte counts as a second, independent step.HL may have an even more detrimental effect on arteriogenesis than DM [[14]van Weel V. de Vries M. Voshol P.J. Verloop R.E. Eilers P.H. van Hinsbergh V.W. et al.Hypercholesterolemia reduces collateral artery growth more dominantly than hyperglycemia or insulin resistance in mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1383-1390Crossref PubMed Scopus (58) Google Scholar]. Therefore, it will be of utmost importance to further investigate the mechanisms hampering monocyte chemotaxis in HL. This should provide important insight into the pathophysiology of HL and into the basis for therapeutic correction of monocyte function to improve arteriogenesis in HL. It is tempting to speculate that the HL-related monocyte defect is different from the DM-related phenotype as monocyte chemokinesis is significantly elevated in HL.This is the first description of monocyte dysfunction in ApoE−/− mice, namely an impaired chemotactic response. This is likely to explain some of the pathogenetic consequences of HL including the activated atherogenesis as well as the hampered angiogenesis/arteriogenesis.Conflict of interestNothing to disclose. Hyperlipidemia (HL) is a major cardiovascular risk factor promoting atherogenesis leading to coronary and peripheral artery disease. Collateral vessel development, a crucial compensatory mechanism in peripheral artery disease, is negatively influenced by HL [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Monocytes and tissue-resident macrophages accelerate both atherogenesis and arteriogenesis by supplying growth factors, cytokines and proteolytic enzymes [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. The decreased monocyte/macrophage accumulation around growing collateral vessels in HL is associated with jeopardized arteriogenesis [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Ligand-induced monocyte extravasation to sites of collateral growth is a crucial step. This involves several factors including monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial factor-A (VEGF-A), which facilitate monocyte chemotaxis in arteriogenesis, a short lasting and well defined process [2Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar, 3Tchaikovski V. Fellbrich G. Waltenberger J. The molecular basis of VEGFR-1 signal transduction pathways in primary human monocytes.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 322-328Crossref PubMed Scopus (87) Google Scholar]. Previous studies in humans indicated that cardiovascular risk factors such as HL severely hamper ex vivo monocyte chemotaxis to VEGF-A and MCP-1 [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar]. Based on these findings, we proposed that monocytes can be used as circulating "biosensors" to detect metabolic deregulation and elevated cardiovascular risk [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar]. However, HL often co-exists with other cardiovascular risk factors such as diabetes mellitus (DM) or smoking, all known to negatively affect monocyte chemotaxis [4Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar, 5Tchaikovski V. Olieslagers S. Bohmer F.D. Waltenberger J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes.Circulation. 2009; 120: 150-159Crossref PubMed Scopus (87) Google Scholar]. We now demonstrate that monocytes from ApoE−/− mice with HL show a completely abrogated chemotaxis towards VEGF-A and a significantly decreased one towards MCP-1. Experiments were approved by the local animal ethical committee of Maastricht University. Blood was sampled [[6]Tchaikovski S.N. van Vlijmen B.J. Rosing J. Tans G. Development of a calibrated automated thrombography based thrombin generation test in mouse plasma.J. Thromb. Haemost. 2007; 5: 2079-2086Crossref PubMed Scopus (53) Google Scholar] and white blood cell counts were performed. Lipid values were measured enzymatically. Plasma lipid levels were significantly elevated in ApoE−/− mice (Table 1). ApoE−/− mice had significantly higher numbers of CD11b + monocytes (p < 0.05). LDL — low density lipoprotein, HDL — high density lipoprotein. For chemotaxis analysis blood from sex, age and genetically (C57Bl/6 or ApoE−/−) matched mice (avg. 5–6 mice per experimental condition, each condition was repeated 3–6 times — "n" in Fig. 1) was pooled and chemotaxis analysis of isolated monocytes was performed [[7]Kerber M. Reiss Y. Wickersheim A. Jugold M. Kiessling F. Heil M. et al.Flt-1 signaling in macrophages promotes glioma growth in vivo.Cancer Res. 2008; 68: 7342-7351Crossref PubMed Scopus (134) Google Scholar]. Monocytes from ApoE−/− mice show significantly impaired chemotactic responses to VEGF-A and MCP-1 compared to wild-type (WT) mice (Fig. 1A, B). In fact, chemotaxis to VEGF-A was not distinguishable from chemokinesis. In contrast, MCP-1-induced chemotaxis was reduced with only a moderate stimulation at the optimal concentration of 10 ng/mL (p < 0.05). Furthermore, monocytes from ApoE−/− mice had a significantly elevated chemokinesis (Fig. 1C) as compared to WT mice (p < 0.05). The pioneering finding of this study is that HL-conditioned monocytes from ApoE−/− mice are dysfunctional, as chemotaxis towards both VEGF-A or MCP-1 is severely impaired. These data imply that i.) monocytes show a functional defect in the presence of HL, and that ii.) monocyte dysfunction is likely to contribute to pathological changes observed in ApoE−/− mice [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Besides stimulation of atherogenesis [[8]Hansson G.K. Hermansson A. The immune system in atherosclerosis.Nat. Immunol. 2011; 12: 204-212Crossref PubMed Scopus (1546) Google Scholar], chronic HL impairs arteriogenesis [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Monocytes contribute to arteriogenesis by VEGFR-1- [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar] or CCR-2-mediated [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar] migration from the blood stream to the growing vessel [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. Reduced expression of arteriogenic factors is an unlikely cause for impaired arteriogenesis in HL as therapeutic rescue attempts with either VEGF-A or MCP-1 largely failed [1Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar, 9van Royen N. Hoefer I. Buschmann I. Kostin S. Voskuil M. Bode C. et al.Effects of local MCP-1 protein therapy on the development of the collateral circulation and atherosclerosis in Watanabe hyperlipidemic rabbits.Cardiovasc. Res. 2003; 57: 178-185Crossref PubMed Scopus (64) Google Scholar]. Furthermore, impaired arteriogenesis in HL mice was accompanied by decreased recruitment of macrophages both under the HL conditions and following the arteriogenic stimulation [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. This previous finding implies – in the light of our novel data – that both native as well as growth factor-stimulated arteriogenesis are reduced in HL due to a reduced/delayed accumulation of dysfunctional blood-derived monocytes at sites of vascular repair [[1]Tirziu D. Moodie K.L. Zhuang Z.W. Singer K. Helisch A. Dunn J.F. et al.Delayed arteriogenesis in hypercholesterolemic mice.Circulation. 2005; 112: 2501-2509Crossref PubMed Scopus (92) Google Scholar]. Indeed, bone marrow-derived cells from WT mice alleviate hindlimb ischemia in ApoE−/− mice by improving blood flow and promoting arteriogenesis [[10]Terry T. Chen Z. Dixon R.A. Vanderslice P. Zoldhelyi P. Willerson J.T. et al.CD34/M-cadherin bone marrow progenitor cells promote arteriogenesis in ischemic hindlimbs of ApoE mice.PLoS One. 2011; 6: e20673Crossref PubMed Scopus (14) Google Scholar]. Our previous work on human monocytes documented an impaired chemotactic response to arteriogenic ligands VEGF-A and MCP-1 [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar] in HL. Our novel findings proof the same in ApoE−/− mice. Impaired monocyte chemotaxis to VEGF-A contributes to impaired arteriogenesis in DM [[4]Waltenberger J. Stress testing at the cellular and molecular level to unravel cellular dysfunction and growth factor signal transduction defects: what Molecular Cell Biology can learn from Cardiology.Thromb. Haemost. 2007; 98: 975-979PubMed Google Scholar]. DM induces unspecific monocyte activation secondary to increased oxidative stress and advanced glycation of functionally relevant molecules [[5]Tchaikovski V. Olieslagers S. Bohmer F.D. Waltenberger J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes.Circulation. 2009; 120: 150-159Crossref PubMed Scopus (87) Google Scholar]. The described monocyte dysfunction in HL may be due to monocyte activation following lipid overload-induced oxidative stress and leads to up-regulation of adhesion molecules [[11]Tabas I. The role of endoplasmic reticulum stress in the progression of atherosclerosis.Circ. Res. 2010; 107: 839-850Crossref PubMed Scopus (364) Google Scholar]. This activation of monocytes may explain the observed elevated chemokinesis in ApoE−/− mice (Fig. 1C) and extravasation to inflamed endothelium of atherosclerotic plaques. Altogether, increased adhesive properties, increased monocyte numbers (Table 1) and extensive intraplaque angiogenesis will promote atherogenesis by progressive monocyte migration to the lesion [[8]Hansson G.K. Hermansson A. The immune system in atherosclerosis.Nat. Immunol. 2011; 12: 204-212Crossref PubMed Scopus (1546) Google Scholar] despite partially impaired migration to pro-atherogenic stimuli (here tested MCP-1). A broad spectrum of chemokines is responsible for monocyte recruitment in atherosclerosis. Modified lipoproteins may serve as chemoattractants [[8]Hansson G.K. Hermansson A. The immune system in atherosclerosis.Nat. Immunol. 2011; 12: 204-212Crossref PubMed Scopus (1546) Google Scholar] as well. Therefore, therapeutic strategies may need to target multiple targets. In the process of compensatory arteriogenesis the stimuli for monocyte recruitment and the therapeutic time window (days/weeks) are rather short compared to atherosclerosis (years/decades) [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. Therefore a decreased monocyte response demonstrated in our study may provide a functional basis for impaired/delayed arteriogenesis while atherogenesis continues. Lowering monocyte numbers can block the progression of atherosclerosis [[12]Swirski F.K. Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure.Science. 2013; 339: 161-166Crossref PubMed Scopus (699) Google Scholar]. The applicability, however, is limited by the fact that post-infarction monocytosis is physiologically required for both healing the damaged myocardium [[13]Dutta P. Courties G. Wei Y. Leuschner F. Gorbatov R. Robbins C.S. et al.Myocardial infarction accelerates atherosclerosis.Nature. 2012; 487: 325-329Crossref PubMed Scopus (727) Google Scholar] and for neovascularisation [[2]Schaper W. Collateral circulation: past and present.Basic Res. Cardiol. 2009; 104: 5-21Crossref PubMed Scopus (305) Google Scholar]. These findings point towards the dilemma of why and how monocytes promote tissue healing in infarcted myocardium and in parallel worsen atherosclerosis. Monocytosis is an accompanying condition featuring several cardiovascular risk factors (e.g., HL, DM) in which the healing capability of monocytes is disturbed. Our findings stress the importance of therapeutically improving monocyte responsiveness to "healing" stimuli such as VEGF, which then could allow to therapeutically lower monocyte counts as a second, independent step. HL may have an even more detrimental effect on arteriogenesis than DM [[14]van Weel V. de Vries M. Voshol P.J. Verloop R.E. Eilers P.H. van Hinsbergh V.W. et al.Hypercholesterolemia reduces collateral artery growth more dominantly than hyperglycemia or insulin resistance in mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1383-1390Crossref PubMed Scopus (58) Google Scholar]. Therefore, it will be of utmost importance to further investigate the mechanisms hampering monocyte chemotaxis in HL. This should provide important insight into the pathophysiology of HL and into the basis for therapeutic correction of monocyte function to improve arteriogenesis in HL. It is tempting to speculate that the HL-related monocyte defect is different from the DM-related phenotype as monocyte chemokinesis is significantly elevated in HL. This is the first description of monocyte dysfunction in ApoE−/− mice, namely an impaired chemotactic response. This is likely to explain some of the pathogenetic consequences of HL including the activated atherogenesis as well as the hampered angiogenesis/arteriogenesis. Conflict of interestNothing to disclose. Nothing to disclose. This study was supported in part by grant from Cells-in-Motion Cluster of Excellence ( EXC 1003—CiM ), University of Münster, Münster, and by grant from the Cardiovascular Research Institute Maastricht ( 30983187N ).
Arteriogenesis
Collateral circulation
Monocyte
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Atherosclerosis is a chronic sterile inflammation of the vascular wall triggered by hyperlipidemia. The role of dendritic cells (DCs) in the development of atherosclerosis has not been recognised until the last decade. DCs can engulf lipids to adopt a foam cell-like appearance that may constitute the earliest stages of plaque formation. DCs may also recruit T cells to the inflamed vessel wall via secretion of chemokines and stimulate T cell responses via cytokines. PPARD is a nuclear receptor, which acts as a sensor of native and oxidized fatty acids. We examined whether PPARD regulates DC function in response to hyperlipidemia and affect atherosclerotic lesion growth. We used Ppard fl/fl mice and Itgax-cre (CD11c-Cre) mice crossed with Apoe -/- mice to generate Ppard f/f ;CD11c Cre/+ ;Apoe -/- as DC-specific knockout of PPARD on Apoe-KO background (Ppard DC-KO ) and Ppard f/f ;Apoe -/- (Ppard DC-WT ) as controls. Ppard DC-KO and Ppard DC-WT were fed with high cholesterol diet for 4 months. Tissues were dissected and digested to obtain single cell suspension for flow cytometric analysis. Bone marrow derived DCs were isolated and cultured in RPMI with serum and GM-CSF and maturated with LPS.Atherosclerotic lesion size and collagen deposition decreased in Ppard DC-KO mice. Less CD4 and CD8 T lymphocytes were found in the atherosclerotic lesion of Ppard DC-KO mice comparing to Ppard DC-WT mice. Ppard deletion in DCs reduced DC infiltration especially CD11b + CD103 - DCs in the atherosclerotic lesion. Production of IFNγ from CD4 + T cells decreased in Ppard DC-KO mice. In BMDCs from Ppard DC-KO mice, LPS and palmitic acid induced expression of co-stimulatory molecules CD80 and CD86, as well as TNF were decreased. LDL uptake was attenuated in BMDCs from PpardDC-KO mice. Our results suggested that PPARD may be involved in DC mediated T cell activation in response to hyperlipidemia. Deletion of PPARD in DCs attenuated plaque formation in atherosclerosis. Whether lipid sensing and uptake by PPARD is required for the enhanced inflammatory response in atherosclerotic mice requires further study. (This study is supported by Hong Kong Health Bureau HMRF 05162906 and 01150057, Hong Kong RGC T402/13-N and CRF C4024-16W, and CUHK Direct grants)
Knockout mouse
CD11c
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Background: IκBNS is a nuclear IκB protein which regulates nuclear factor-κB dependent interleukin (IL) -6 production. Although IL-6 is an important biomarker for cardiovascular diseases, the role of IκBNS in the development of atherosclerosis is poorly understood. Methods and Results: Mice that lacked IκBNS (IκBNS-/-) were crossed with LDL receptor-deficient (LDLr-/-) mice and formation of atherosclerotic lesion was analyzed after 16 weeks consumption of a high-fat diet. When compared with single LDLr-/- mice (n=6), the surface atherosclerotic lesions in aortas were significantly increased in IκBNS-/-LDLr-/- mice (n=6) (22.7 ± 3.0% vs. 6.2 ± 1.4 %; p <0.001)(Figure). Immunostaining revealed significant increase in both Mac-3-positive (1.6-fold, p <0.05) and α-SMA-positive (3.6-fold, p <0.01) lesions in IκBNS-/-LDLr-/- mice compared with LDLr-/- mice. Furthermore, IL-6 expression (2.2-fold, p<0.001), and percent phospho-STAT3-positive cell (1.9-fold, p<0.05) were increased in IκBNS-/-LDLr-/- mice, indicating IκBNS deficiency promoted proliferation of both macrophage and smooth muscle cells via the IL-6-Stat3 signaling pathway. Then, we studied the function of macrophages. After LPS stimulation, the mRNA levels of both IL-6 (2.4-fold, p<0.001) and IL-18 (1.4-fold, p<0.05) were increased in the macrophages from IκBNS-/-LDLr-/- mice compared with LDLr-/- mice. Moreover, ELISA experiments revealed IκBNS-/-LDLr-/- macrophages produce much higher level of IL-6 in culture supernatants compared with LDL-/- macrophages (1.9-fold, p<0.01). Conclusions: The present study shows that IκBNS gene deficiency in atherogenic mice led to the increase of IL-6 production and the development of atherosclerosis.
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