The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, the mechanism by which this occurs is not clearly defined. Here, we tested in mice the hypothesis that CD36, a class B scavenger receptor expressed on macrophages, has a role in this process. Using both in vivo and in vitro migration assays, we found that oxidized LDL (oxLDL), but not native LDL, inhibited migration of WT mouse macrophages but not CD36-deficient cells. We further observed a crucial role for CD36 in modulating the in vitro migratory response of human peripheral blood monocyte-derived macrophages to oxLDL. oxLDL also induced rapid spreading and actin polymerization in CD36-sufficient but not CD36-deficient mouse macrophages in vitro. The underlying mechanism was dependent on oxLDL-mediated CD36 signaling, which resulted in sustained activation of focal adhesion kinase (FAK) and inactivation of Src homology 2-containing phosphotyrosine phosphatase (SHP-2). The latter was due to NADPH oxidase-mediated ROS generation, resulting in oxidative inactivation of critical cysteine residues in the SHP-2-active site. Macrophage migration in the presence of oxLDL was restored by both antioxidants and NADPH oxidase inhibitors, which restored the dynamic activation of FAK. We conclude therefore that CD36 signaling in response to oxLDL alters cytoskeletal dynamics to enhance macrophage spreading, inhibiting migration. This may induce trapping of macrophages in the arterial intima and promote atherosclerosis.
Activated platelets release biologically active compounds, which then recruit additional platelets into an evolving thrombus. We studied activation of alpha(IIb)beta(3) and exposure of P-selectin on platelets recruited by releasates obtained from collagen-treated platelets and evaluated modifications in prothrombotic effects of releasates induced by platelet-erythrocyte interactions and aspirin treatment. Releasates from collagen-stimulated platelets induced alpha(IIb)beta(3) activation and P-selectin exposure (monitored by flow cytometry using fluorescein isothiocyanate-PAC-1 and phycoerythrin-CD62 antibodies). These responses were markedly amplified by releasates from combined platelet-erythrocyte suspensions. This finding demonstrates a novel mechanism(s) by which erythrocytes intensify platelet aggregability and mediate increased platelet recruitment. Because P-selectin and alpha(IIb)beta(3) are potential sites for platelet-leukocyte interactions, erythrocytes may also modulate leukocyte recruitment. Following aspirin ingestion both the recruiting capacity of platelet releasates and erythrocyte-induced amplification of platelet recruitment were down-regulated. These events represent an additional antithrombotic property of aspirin. We also examined the possibility that arachidonic acid, or eicosanoids derived therefrom, can induce a prothrombotic activity of erythrocytes. The TXA(2)-analog U46 619 and free arachidonate, but not PGI(2) or 12-HETE, induced increases in cytosolic Ca(++) and promoted phosphatidylserine (PS) exposure on a subpopulation of erythrocytes. PS exposure and increases in erythrocyte [Ca(++)](i) are associated with enhanced procoagulant activity, increased endothelial adhesion, and reduced erythrocyte deformability. Our findings, therefore, suggest that TXA(2) and arachidonic acid, derived from activated platelets, induce a prothrombotic phenotype on erythrocytes in proximity. We conclude that by these mechanisms, erythrocytes can actively contribute to platelet-driven thrombogenesis and microvascular occlusion.
To determine a role for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) in regulation of human platelet reactivity by human endothelial cells (EC), we studied combined suspensions of human umbilical vein endothelial cells (HU-VEC, passage 2 through 3) and washed human platelets. Confluent HUVEC monolayers were treated with aspirin (1 mmol/L) to prevent prostacyclin (PGI2) formation, washed, and harvested. Aspirin-treated platelets alone (58 x 10(6)) were fully aggregated by thrombin at 0.05 U/mL or more. In the presence of 10(6) HUVEC, however, platelet serotonin release and aggregation in response to thrombin at doses as high as 0.5 U/mL were blocked. We demonstrated for the first time that inhibition of aggregation and serotonin release, due to EDRF/NO, occurred in parallel. HUVEC-dependent inhibition of platelet responsiveness was enhanced by superoxide dismutase (SOD) and reversed by hemoglobin. The inhibitory effect was also reversed by preincubation of HUVEC with NG-monomethyl-L-arginine (NMA) or NG-nitro-L-arginine (NNA) through competitive blockade of arginine metabolism. Pretreatment of platelets with methylene blue indicated that EC-dependent inhibition of platelet reactivity occurred through activation of platelet soluble guanylate cyclase. When platelets and HUVEC were separated by a permeable membrane and both cells were stimulated by thrombin, platelets remained unresponsive. This indicated that inhibition was induced by a fluid-phase mediator, independent of direct cell-cell contact. These data demonstrate that EDRF/NO formation from L-arginine by human EC plays an important role as an aspirin-insensitive fluid-phase inhibitor of human platelet reactivity.
Cerebral venous thrombosis (CVT) may be a manifestation of underlying autoimmune disease. Antibodies against annexin A2 (anti-A2Ab) coincide with antiphospholipid syndrome, in which antiphospholipid antibodies (aPLA) are associated with thrombosis in any vascular bed. Annexin A2, a profibrinolytic receptor and binding site for β2-glycoprotein-I, the main target for aPLA, is highly expressed on cerebral endothelium. Here we evaluate the prevalence of anti-A2Ab in CVT.
Forty individuals with objectively documented CVT (33 women and 7 men) and 145 healthy controls were prospectively studied for hereditary and acquired prothrombotic risk factors, classical aPLA, and anti-A2Ab.
One or more prothrombotic risk factors were found in 85% of CVT subjects, (pregnancy/puerperium in 57.5%, classical aPLA in 22.5%, and hereditary procoagulant risk factors in 17.5%). Anti-A2Ab (titer >3 SD) were significantly more prevalent in patients with CVT (12.5%) than in healthy individuals (2.1%, P<0.01, OR, 5.9).
Anti-A2Ab are significantly associated with CVT and may define a subset of individuals with immune-mediated cerebral thrombosis.
Cigarette smoking is a major risk factor for atherosclerosis and cardiovascular disease. CD36 mediates oxidized LDL (oxLDL) uptake and contributes to macrophage foam cell formation. We investigated a role for the CD36 pathway in nicotine-induced activation of macrophages and foam cell formation in vitro and in vivo. Nicotine in the same plasma concentration range found in smokers increased the CD36(+)/CD14(+) cell population in human peripheral blood mononuclear cells, increased CD36 expression of human THP1 macrophages, and increased macrophage production of reactive oxygen species, PKCδ phosphorylation, and peroxisome proliferator-activated receptor-γ (PPARγ) expression. Nicotine-induced CD36 expression was suppressed by antioxidants and by specific PKCδ and PPARγ inhibitors, implicating mechanistic roles for these intermediates. Nicotine synergized with oxLDL to increase macrophage expression of CD36 and cytokines TNF-α, monocyte chemoattractant protein-1, IL-6, and CXCL9, all of which were prevented by CD36 small interfering (si)RNA. Incubation with oxLDL (50 μg/ml) for 72 h resulted in lipid deposition in macrophages and foam cell formation. Preincubation with nicotine further increased oxLDL-induced lipid accumulation and foam cell formation, which was also prevented by CD36 siRNA. Treatment of apoE-/- mice with nicotine markedly exacerbated inflammatory monocyte levels and atherosclerotic plaque accumulation, effects that were not seen in CD36-/- apoE-/- mice. Our results show that physiological levels of nicotine increase CD36 expression in macrophages, a pathway that may account at least in part for the known proinflammatory and proatherogenic properties of nicotine. These results identify such enhanced CD36 expression as a novel nicotine-mediated pathway that may constitute an independent risk factor for atherosclerosis in smokers. The results also suggest that exacerbated atherogenesis by this pathway may be an adverse side effect of extended use of high concentrations of nicotine independent of their mode of administration.
Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.
Annexin A2 is a multicompartmental protein that orchestrates a spectrum of dynamic membrane-related events. At cell surfaces, A2 forms the (A2•S100A10)2 complex which accelerates tissue plasminogen activator-dependent activation of the fibrinolytic protease, plasmin. Anti-A2 antibodies are associated with clinical thrombosis in antiphospholipid syndrome, whereas overexpression of A2 promotes hyperfibrinolytic bleeding in acute promyelocytic leukemia. A2 is upregulated in hypoxic tissues, and mice deficient in A2 are resistant to hypoxia-related retinal neovascularization in a model of diabetic retinopathy. Within the cell, A2 regulates membrane fusion processes involved in the secretion of pre-packaged, ultra-large molecules. In stimulated dendritic cells, A2 maintains lysosomal membrane integrity, thereby modulating inflammasome activation and cytokine secretion. Together, these findings suggest an emerging, multifaceted role for annexin A2 in human health and disease. The author's work has been inspired by numerous colleagues and mentors, and by the author's grandfather, and former ACCA member, Dr. J. Burns Amberson.