Akm Khyrul Wara

Kruppel-like Factor 4 Is a Mediator of Proinflammatory Signaling in Macrophages

Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-γ (IFN-γ), lipopolysaccharide (LPS), or tumor necrosis factor-α can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-β1 (TGF-β1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-γ, LPS, or tumor necrosis factor-α and decreased by TGF-β1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-β1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-γ, LPS, or IFN-γ plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-β1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at –95 and –212 bp, and mutation of these sites diminished induction by IFN-γ and LPS. We further provide evidence that KLF4 interacts with the NF-κB family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-β1/Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.

The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation

Monocyte differentiation involves the participation of lineage‐restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel‐like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte‐restricted and stage‐specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL‐60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester‐induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1–/– fetal liver cells along the monocytic lineage and can activate the monocytic‐specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation.

Adiponectin Inhibits the Production of CXC Receptor 3 Chemokine Ligands in Macrophages and Reduces T-Lymphocyte Recruitment in Atherogenesis

Obese individuals often have low plasma adiponectin and concomitant chronic inflammation with a predisposition to metabolic and cardiovascular diseases. The present study reports a novel antiinflammatory action of adiponectin in human monocyte-derived macrophages (M&PHgr;) suppressing T-lymphocyte accumulation in atherogenesis. RNA profiling of lipopolysaccharide-stimulated human M&PHgr; identified CXC chemokine ligands (CXCLs), such as IP-10 (interferon [IFN]-inducible protein 10) (CXCL10), I-TAC (IFN-inducible T-cell α chemoattractant) (CXCL11), and Mig (monokine induced by IFN-γ) (CXCL9), T-lymphocyte chemoattractants associated with atherogenesis, among the top 14 transcripts suppressed by adiponectin. Real-time quantitative RT-PCR and ELISA verified that adiponectin inhibited expression of these chemokines at both the mRNA and protein levels in a concentration-dependent manner. Adiponectin reduced the release by lipopolysaccharide-stimulated M&PHgr; of chemoattractant activity for CXC chemokine receptor 3–transfected (receptor for IP-10, Mig, and I-TAC) lymphocytes. Adiponectin decreased lipopolysaccharide-inducible IP-10 promoter activity in promoter-transfected THP-1 M&PHgr; but did not change IP-10 mRNA stability. In lipopolysaccharide-stimulated M&PHgr;, reduction of IFN-β by adiponectin preceded inhibition of IP-10 mRNA expression. Immunoblot and chromatin immunoprecipitation analyses demonstrated that adiponectin attenuated activation of the transcription factor IFN regulatory factor 3, involved in the MyD88-independent pathway of Toll-like receptor 4 signaling, and subsequent IFN regulatory factor 3 binding to IFN-β promoter. In vivo studies further demonstrated that apolipoprotein E/adiponectin double-deficient (apoE −/−APN−/−) mice had increased plasma IP-10 levels, accelerated T-lymphocyte accumulation in atheromata, and augmented atherogenesis compared with apoE single-deficient (apoE−/−APN+/+) mice. This study establishes that low levels of adiponectin associated with obesity, the metabolic syndrome, and diabetes favor T-lymphocyte recruitment and contribute to adaptive immune response during atherogenesis.

Systemic Delivery of MicroRNA-181b Inhibits Nuclear Factor-κB Activation, Vascular Inflammation, and Atherosclerosis in Apolipoprotein E–Deficient Mice

Rationale: Activated nuclear factor (NF)-&kgr;B signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-&kgr;B may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-&kgr;B signaling and effects on atherosclerosis. Methods and Results: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-&kgr;B signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-&kgr;B-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-&agr;3, an effect that reduced NF-&kgr;B nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-&kgr;B signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-&kgr;B nuclear translocation in leukocytes does not involve importin-&agr;3, but rather importin-&agr;5, which miR-181b does not target, highlighting that inhibition of NF-&kgr;B signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-&kgr;B and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

Systemic Delivery of MicroRNA-181b Inhibits NF-κB Activation, Vascular Inflammation, and Atherosclerosis in Apoe-/- Mice

Rationale: Activated nuclear factor (NF)-&kgr;B signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-&kgr;B may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-&kgr;B signaling and effects on atherosclerosis. Methods and Results: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-&kgr;B signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-&kgr;B-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-&agr;3, an effect that reduced NF-&kgr;B nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-&kgr;B signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-&kgr;B nuclear translocation in leukocytes does not involve importin-&agr;3, but rather importin-&agr;5, which miR-181b does not target, highlighting that inhibition of NF-&kgr;B signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-&kgr;B and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

MicroRNA-26a regulates pathological and physiological angiogenesis by targeting BMP/SMAD1 signaling

Rationale: The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. Objective: We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. Methods and Results: We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3′-untranslated region, an effect that decreased expression of Id1 and increased p21WAF/CIP and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti–miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. Conclusions: These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Role of Krüppel-like factors in leukocyte development, function, and disease

The Krüppel-like transcription factor (KLF) family participates in diverse aspects of cellular growth, development, differentiation, and activation. Recently, several groups have identified new connections between the function of these factors and leukocyte responses in health and disease. Gene targeting of individual KLFs in mice has uncovered novel and unexpected physiologic roles among myeloid and lymphocyte cell lineage maturation, particularly in the bone marrow niche and blood. In addition, several KLF family members are downstream targets of stimuli and signaling pathways critical to T-cell trafficking, T regulatory cell differentiation or suppressor function, monocyte/macrophage activation or renewal, and B memory cell maturation or activation. Indeed, KLFs have been implicated in subtypes of leukemia, lymphoma, autoimmunity, and in acute and chronic inflammatory disease states, such as atherosclerosis, diabetes, and airway inflammation, raising the possibility that KLFs and their upstream signals are of therapeutic interest. This review focuses on the relevant literature of Krüppel-like factors in leukocyte biology and their implications in clinical settings.

Kruppel-like Factor KLF10 Targets Transforming Growth Factor-β1 to Regulate CD4+CD25− T Cells and T Regulatory Cells

CD4+CD25+ regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4+CD25− T cell activation through distinct mechanisms involving transforming growth factor (TGF)-β1 and Foxp3. KLF10 overexpressing CD4+CD25− T cells induced both TGF-β1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10−/− CD4+CD25− T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10−/− CD4+CD25− T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10−/− T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-β1, an effect completely rescued by exogenous treatment with TGF-β1. Mechanistic studies demonstrate that in response to TGF-β1, KLF10 can transactivate both TGF-β1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10−/− CD4+CD25− T cells promoted atherosclerosis by ∼2-fold in ApoE−/−/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-β1 in both CD4+CD25− T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

Bone marrow-derived CMPs and GMPs represent highly functional proangiogenic cells: Implications for ischemic cardiovascular disease

Clinical studies using bone marrow-derived proangiogenic cells (PACs) have demonstrated modest improvements of function and/or perfusion of ischemic myocardium or skeletal muscle. Because the identities of these PACs and their functional ability to promote neovascularization remain poorly understood, it is possible that a subset of robust PACs exists but is obscured by the heterogeneous nature of this cell population. Herein, we found that common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) preferentially differentiate into PACs compared with megakaryocyte-erythrocyte progenitors, hematopoietic stem cells, and common lymphoid progenitors. In vivo hindlimb ischemia studies and Matrigel plug assays verified the enhanced neovascularization properties uniquely associated with PACs derived from CMPs and GMPs. Taken together, these observations identify CMPs and GMPs as key bone marrow progenitors for optimal PAC function in vitro and in vivo and provide a foundation for novel therapeutic approaches to modulate angiogenesis.

TGF-β1 signaling and Krüppel-like factor 10 regulate bone marrow–derived proangiogenic cell differentiation, function, and neovascularization

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-β1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ∼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-β responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-β1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-β1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.