Xinghui Sun

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.

IgE stimulates human and mouse arterial cell apoptosis and cytokine expression and promotes atherogenesis in Apoe–/– mice

IgE has a key role in the pathogenesis of allergic responses through its ability to activate mast cells via the receptor FcεR1. In addition to mast cells, many cell types implicated in atherogenesis express FcεR1, but whether IgE has a role in this disease has not been determined. Here, we demonstrate that serum IgE levels are elevated in patients with myocardial infarction or unstable angina pectoris. We found that IgE and the FcεR1 subunit FcεR1α were present in human atherosclerotic lesions and that they localized particularly to macrophage-rich areas. In mice, absence of FcεR1α reduced inflammation and apoptosis in atherosclerotic plaques and reduced the burden of disease. In cultured macrophages, the presence of TLR4 was required for FcεR1 activity. IgE stimulated the interaction between FcεR1 and TLR4, thereby inducing macrophage signal transduction, inflammatory molecule expression, and apoptosis. These IgE activities were reduced in the absence of FcεR1 or TLR4. Furthermore, IgE activated macrophages by enhancing Na+/H+ exchanger 1 (NHE1) activity. Inactivation of NHE1 blocked IgE-induced macrophage production of inflammatory molecules and apoptosis. Cultured human aortic SMCs (HuSMCs) and ECs also exhibited IgE-induced signal transduction, cytokine expression, and apoptosis. In human atherosclerotic lesions, SMCs and ECs colocalized with IgE and TUNEL staining. This study reveals what we believe to be several previously unrecognized IgE activities that affect arterial cell biology and likely other IgE-associated pathologies in human diseases.

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.

Endothelial MicroRNAs and Atherosclerosis

The vascular endothelium, a thin layer of endothelial cells (ECs) that line the inner surface of blood vessels, is a critical interface between blood and all tissues. EC activation, dysfunction, and vascular inflammation occur when the endothelium is exposed to various insults such as proinflammatory cytokines, oxidative stress, hypertension, hyperglycemia, aging, and shear stress. These insults lead to the pathogenesis of a range of disease states, including atherosclerosis. Several signaling pathways, especially nuclear factor κB mediated signaling, play crucial roles in these pathophysiological processes. Recently, microRNAs (miRNAs) have emerged as important regulators of EC function by fine-tuning gene expression. In this review, we discuss how miRNAs regulate EC function and vascular inflammation in response to a variety of pathophysiologic stimuli. An understanding of the role of miRNAs in EC activation and dysfunction may provide novel targets and therapeutic opportunities for controlling atherosclerosis and other chronic inflammatory disease states.

Role of miR-181 family in regulating vascular inflammation and immunity

The microRNA family, miR-181, plays diverse roles in regulating key aspects of cellular growth, development, and activation. Accumulating evidence supports a central role for the miR-181 family in vascular inflammation by controlling critical signaling pathways, such as downstream NF-κB signaling, and targets relevant to endothelial cell activation and immune cell homeostasis. This review examines the current knowledge of the miR-181 familys role in key cell types that critically control cardiovascular inflammation under pathological and physiological stimuli.

miRNAs in atherosclerotic plaque initiation, progression, and rupture

Atherosclerosis is a chronic immune-inflammatory disorder that integrates multiple cell types and a diverse set of inflammatory mediators. miRNAs are emerging as important post-transcriptional regulators of gene expression in most, if not all, vertebrate cells, and constitute central players in many physiological and pathological processes. Rapidly accumulating experimental studies reveal their key role in cellular and molecular processes related to the development of atherosclerosis. We review current evidence for the involvement of miRNAs in early atherosclerotic lesion formation and in plaque rupture and erosion. We conclude with a perspective on the clinical relevance, therapeutic opportunities, and future challenges of miRNA biology in understanding the pathogenesis of this complex disease.

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.