Yunmei Wang

Platelet Expression Profiling and Clinical Validation of Myeloid-Related Protein-14 as a Novel Determinant of Cardiovascular Events

Background— Platelets participate in events that immediately precede acute myocardial infarction. Because platelets lack nuclear DNA but retain megakaryocyte-derived mRNAs, the platelet transcriptome provides a novel window on gene expression preceding acute coronary events. Methods and Results— We profiled platelet mRNA from patients with acute ST-segment–elevation myocardial infarction (STEMI, n=16) or stable coronary artery disease (n=44). The platelet transcriptomes were analyzed and single-gene models constructed to identify candidate genes with differential expression. We validated 1 candidate gene product by performing a prospective, nested case-control study (n=255 case-control pairs) among apparently healthy women to assess the risk of future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) associated with baseline plasma levels of the candidate protein. Platelets isolated from STEMI and coronary artery disease patients contained 54 differentially expressed transcripts. The strongest discriminators of STEMI in the microarrays were CD69 (odds ratio 6.2, P<0.001) and myeloid-related protein-14 (MRP-14; odds ratio 3.3, P=0.002). Plasma levels of MRP-8/14 heterodimer were higher in STEMI patients (17.0 versus 8.0 &mgr;g/mL, P<0.001). In the validation study, the risk of a first cardiovascular event increased with each increasing quartile of MRP-8/14 (Ptrend<0.001) such that women with the highest levels had a 3.8-fold increase in risk of any vascular event (P<0.001). Risks were independent of standard risk factors and C-reactive protein. Conclusions— The platelet transcriptome reveals quantitative differences between acute and stable coronary artery disease. MRP-14 expression increases before STEMI, and increasing plasma concentrations of MRP-8/14 among healthy individuals predict the risk of future cardiovascular events.

Myeloid-Related Protein-8/14 Is Critical for the Biological Response to Vascular Injury

Background— Myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) are members of the S100 family of calcium-modulated proteins that regulate myeloid cell function and control inflammation, in part, through activation of Toll-like receptor-4 and the receptor for advanced glycation end products. A transcriptional profiling approach in patients with acute coronary syndromes identified MRP-14 as a novel predictor of myocardial infarction. Further studies demonstrated that elevated plasma levels of MRP-8/14 heterodimer predict increased risk of first and recurrent cardiovascular events. Beyond its serving as a risk marker, whether MRP-8/14 participates directly in vascular inflammation and disease remains unclear. Methods and Results— We evaluated vascular inflammation in wild-type and MRP-14–deficient (MRP-14−/−) mice that lack MRP-8/14 complexes with experimental arterial injury, vasculitis, or atherosclerosis. After femoral artery wire injury, MRP-14−/− mice had significant reductions in leukocyte accumulation, cellular proliferation, and neointimal formation compared with wild-type mice. In a cytokine-induced local Shwartzman-like reaction that produces thrombohemorrhagic vasculitis, MRP-14−/− mice had significant reductions in neutrophil accumulation, lesion severity, and hemorrhagic area. In response to high-fat feeding, mice doubly deficient in apolipoprotein E and MRP-8/14 complexes had attenuation in atherosclerotic lesion area and in macrophage accumulation in plaques compared with mice deficient in apolipoprotein E alone. Conclusion— This study demonstrates that MRP-8/14 broadly regulates vascular inflammation and contributes to the biological response to vascular injury by promoting leukocyte recruitment.

Inhibition of T Cell Costimulation Abrogates Airway Hyperresponsiveness in a Murine Model

Activation of naive T cells requires at least two signals. In addition to the well characterized interaction of the T cell antigen receptor with the antigen/MHC expressed on an antigen-presenting cell, T cell activation also requires costimulation by a second set of signals. The best characterized costimulatory receptor is CD28, which binds to a family of B7 ligands expressed on antigen-presenting cells. In asthma, although activated T cells play a role in the initiation and maintenance of airway inflammation, the importance of T cell costimulation in bronchial hyperresponsiveness had not been characterized. Therefore, we tested the hypothesis that inhibition of the CD28:B7 costimulatory pathway would abrogate airway hyperresponsiveness. Our results show that blockade of costimulation with CTLA4-Ig, a fusion protein known to prevent costimulation by blocking CD28:B7 interactions, inhibits airway hyperresponsiveness, inflammatory infiltration, expansion of thoracic lymphocytes, and allergen-specific responsiveness of thoracic T cells in this murine model of allergic asthma.

Leukocyte Engagement of Platelet Glycoprotein Ibα via the Integrin Mac-1 Is Critical for the Biological Response to Vascular Injury

Background— Leukocyte-platelet interactions are critical in the initiation and progression of atherosclerosis as well as restenosis. Although the leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) has been implicated in the firm adhesion and transmigration of leukocytes at sites of platelet deposition, the precise αMβ2 counterligand responsible for mediating adhesion-strengthening interactions between neutrophils and platelets in vivo has not previously been identified. Methods and Results— Our previous studies have established the P201-K217 sequence in the αMI domain as the binding site for platelet glycoprotein (GP) Ibα. Here we report that antibody targeting of αM(P201-K217) reduced αMβ2-dependent adhesion to GP Ibα but not other αMβ2 ligands, including fibrinogen, intercellular adhesion molecule-1, and junctional adhesion molecule-3. Anti-αM(P201-K217) inhibited the firm adhesion of both human and murine leukocytes to adherent platelets under laminar flow conditions. In a mouse femoral artery wire injury model, antibody targeting of αM(P201-K217) reduced leukocyte accumulation after injury that was accompanied by inhibition of cellular proliferation and neointimal thickening. Conclusions— This study demonstrates that GP Ibα is a physiologically relevant ligand for αMβ2 and that integrin engagement of GP Ibα is critical to leukocyte function and the biological response to vascular injury. These observations establish a molecular target for selectively disrupting leukocyte-platelet complexes that promote inflammation in thrombosis and restenosis.

Hemizygous deficiency of Krüppel-like factor 2 augments experimental atherosclerosis.

Krüppel-like factor (KLF)2 is a central regulator of endothelial and monocyte/macrophage gene expression and function in vitro. Although the composite effects of KLF2 in these 2 cell types predict that it likely inhibits vascular inflammation, the role of KLF2 in this process in vivo is uncharacterized. In this study, we provide evidence that hemizygous deficiency of KLF2 increased diet-induced atherosclerosis in apolipoprotein E–deficient mice. Our studies highlight an important role for KLF2 in primary macrophage foam cell formation via the potential regulation of the key lipid binding protein adipocyte protein 2/fatty acid–binding protein 4. These novel observations establish that KLF2 is an atheroprotective factor.

Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal.

Platelets Contribute to the Pathogenesis of Experimental Autoimmune Encephalomyelitis

Rationale: Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. Objective: We addressed the role of platelets in mediating CNS inflammation in EAE. Methods and Results: We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control nondiseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression on platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ib&agr; (GPIb&agr;) promotes both platelet adhesion and inflammatory actions of platelets and targeting of GPIb&agr; attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), also reduced EAE severity in mice. Conclusions: Platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.

Down-regulation of the forkhead transcription factor Foxp1 is required for monocyte differentiation and macrophage function.

Down-regulation of the forkhead transcription factor Foxp1 by integrin engagement controls monocyte differentiation in vitro. To determine whether Foxp1 plays a critical role in monocyte differentiation and macrophage functions in vivo, we generated transgenic mice (macFoxp1tg) overexpressing human FOXP1 in monocyte/macrophage lineage cells using the CD68 promoter. Circulating blood monocytes from macFoxp1tg mice have reduced expression of the receptor for macrophage colony-stimulating factor (c-Fms/M-CSFR), impaired migratory capacity, and diminished accumulation as splenic macrophages. Macrophage functions, including cytokine production, phagocytosis, and respiratory burst were globally impaired in macFoxp1tg compared with wild-type cells. Osteoclastogenesis and bone resorption activity were also attenuated in macFoxp1tg mice. In models of chemical and bacterial peritonitis, macFoxp1tg mice exhibited reduced macrophage accumulation, bacterial clearance, and survival. Enforced overexpression of c-Fms/M-CSFR reversed the cytokine production and phagocytosis defects in macFoxp1tg macrophages, indicating that repression of c-fms/M-CSFR is likely the dominant mechanism responsible for Foxp1 action in monocyte differentiation and macrophage function. Taken together, these observations identify down-regulation of Foxp1 as critical for monocyte differentiation and macrophage functions in vivo.

Chronic Skin-Specific Inflammation Promotes Vascular Inflammation and Thrombosis

Patients with psoriasis have systemic and vascular inflammation and are at increased risk for myocardial infarction, stroke, and cardiovascular death. However, the underlying mechanism(s) mediating the link between psoriasis and vascular disease is incompletely defined. This study sought to determine whether chronic skin-specific inflammation has the capacity to promote vascular inflammation and thrombosis. Using the KC-Tie2 doxycycline-repressible (Dox-off) murine model of psoriasiform skin disease, spontaneous aortic root inflammation was observed in 33% of KC-Tie2 compared to 0% of control mice by 12 months of age (P=0.04) and was characterized by the accumulation of macrophages, T-lymphocytes and B-lymphocytes and reduced collagen content and increased elastin breaks. Importantly, aortic inflammation was preceded by increases in serum TNF-α, IL-17A, VEGF, IL-12, MCP-1 and S100A8/A9 as well as splenic and circulating CD11b+Ly-6Chi pro-inflammatory monocytes. Doxycycline treatment of old mice with severe skin disease eliminated skin inflammation and aortic root lesion presence in 1 year old KC-Tie2 animals. Given the bi-directional link between inflammation and thrombosis, arterial thrombosis was assessed in KC-Tie2 and control mice; mean time to occlusive thrombus formation was shortened by 64% (P=0.002) in KC-Tie2 animals; doxycycline treatment returned thrombosis clotting times to control mouse levels (P=0.69). These findings demonstrate that sustained skin-specific inflammation promotes aortic root inflammation and thrombosis and suggest that aggressive treatment of skin inflammation may attenuate pro-inflammatory and prothrombotic pathways that produce cardiovascular disease in psoriasis patients.

Mac-1 (CD11b/CD18) Links Inflammation and Thrombosis After Glomerular Injury

Background— Inflammation and thrombosis coexist in several disorders. Although it is recognized that leukocytes may induce a procoagulant state at sites of inflammation, the critical molecular determinants of this process remain largely unknown. Methods and Results— To examine mechanisms of inflammation-induced thrombosis, we developed a murine model of thrombotic glomerulonephritis (TGN), a known cause of acute renal failure in patients. This model, induced by lipopolysaccharide and antibody to the glomerular basement membrane, led to rapid glomerular neutrophil recruitment, thrombotic glomerular lesions with endothelial cell injury, and renal dysfunction. In mice immunodepleted of neutrophils or lacking the leukocyte-specific integrin Mac-1, neutrophil recruitment, endothelial injury, glomerular thrombosis, and acute renal failure were markedly attenuated despite the robust generation of renal cytokines. Neutrophil elastase is a likely effector of Mac-1 because its activity was reduced in Mac-1–deficient mice and the phenotype in mice deficient in Mac-1 or neutrophil elastase was similar. Platelets accumulated in glomerular capillaries within 4 hours of TGN before evidence of thrombosis. Platelet immunodepletion before TGN markedly exacerbated hematuria (hemorrhage), inflammation, and injury, whereas thrombocytopenic Mac-1–deficient mice remained resistant to disease, indicating that initial glomerular platelet deposition protects the vessel wall from neutrophil-mediated sequelae. The subsequent thrombosis relied on the interaction of Mac-1 on recruited neutrophils with glycoprotein Ibα on platelets as antibody-mediated disruption of this interaction attenuated TGN without affecting renal neutrophil accumulation. Conclusions— These observations establish Mac-1 on neutrophils as a critical molecular link between inflammation and thrombosis and suggest it as an attractive target for antithrombotic therapy.