CD1d serves as a surface receptor for oxidized cholesterol induction of peroxisome proliferator-activated receptor-γ.

OBJECTIVE: The cluster of differentiation-1d (CD1d) recognizes and presents the lipid antigens to NK-T lymphocytes. Atherosclerotic lesions contain atherogenic lipids, mainly cholesterol and its oxides. Peroxisome proliferator-activated receptor-γ (PPARγ) is also known to exist in atherosclerotic lesions, participating in regulation of lipid metabolism. The current study tested whether CD1d acts as a surface receptor that mediates induction and activation of PPARγ by oxysterols commonly found in atherosclerotic lesions. METHODS AND RESULTS: CD1d overexpression in HEK 293 cells transfected with CD1d cDNA was confirmed by fluorescence, flow cytometry, Western blotting and mRNA expression. Tritiated ((3)H) 7-ketocholesterol (7K) was used for lipid binding assays. Radioactive assessment demonstrated an increased 7K-binding activity HEK 293 cells with CD1d overexpression. The 7K binding could be blocked by another oxysterol, 25-hydroxycholesterol, but not by native free cholesterol. Addition of CD1d:IgG dimer protein or an anti-CD1d antibody, but not control IgG, significantly diminished 7K binding to CD1d-expressing HEK 293 cells. CD1d deficiency markedly diminished the 7K-binding in macrophages and smooth muscle cells. Western blot and gel shift assays demonstrated that CD1d-mediated 7K binding induced expression and activation of PPARγ. The PPARγ agonist PGJ2 enhances the 7K stimulatory effect on PPARγ expression and activity but the antagonist GW9662 inhibits the 7K effect on the CD1d-expressing cells. CONCLUSIONS: CD1d acts as a cell surface receptor that recognizes and binds oxysterols and initializes a pathway connecting oxysterol binding to PPARγ activation.

Serum opacity factor enhances HDL-mediated cholesterol efflux, esterification and anti inflammatory effects.

Serum opacity factor (SOF) is a streptococcal protein that disrupts the structure of human high density lipoproteins (HDL) releasing lipid-free apo A-I while forming a large cholesteryl ester-rich particle and a small neo HDL. Given its low cholesterol and high phospholipid contents, we tested the hypotheses that neo HDL is a better substrate for cholesterol esterification via lecithin:cholesterol acyltransferase (LCAT), better than HDL as an acceptor of THP-1 macrophage cholesterol efflux, and improves reduction of oxidized LDL-induced production of inflammatory markers. We observed that both cholesterol efflux and esterification were improved by recombinant (r)SOF treatment of whole plasma and that the underlying cause of the improved cholesterol esterification in plasma and macrophage cholesterol efflux to rSOF-treated plasma was due to the rSOF-mediated conversion of HDL to neo HDL. Moreover, the reduction of secretion of TNF-α and IL-6 by THP-1 cells by neo HDL was twice that of HDL. Studies in BHK cells overexpressing cholesterol transporters showed that efflux to neo HDL occurred primarily via ABCA1 not ABCG1. Thus, rSOF improves two steps in reverse cholesterol transport with a concomitant reduction in the release of macrophage markers of inflammation. We conclude that rSOF catalyzes a novel reaction that might be developed as a new therapy that prevents or reverses atherosclerosis via improved reverse cholesterol transport.

Mediation of electronegative low-density lipoprotein signaling by LOX-1: a possible mechanism of endothelial apoptosis.

The lectin-like oxidized LDL receptor LOX-1 mediates endothelial cell (EC) uptake of experimentally prepared copper-oxidized LDL (oxLDL). To confirm the atherogenic role of this receptor cloned against copper-oxLDL, we examined whether it mediates EC uptake of L5, an electronegative LDL abundant in dyslipidemic but not normolipidemic human plasma. Hypercholesterolemic (LDL-cholesterol, >160 mg/dL) human LDL was fractionated into L1-L5, increasingly electronegative, by ion-exchange chromatography. In cultured bovine aortic ECs (BAECs), L5 upregulated LOX-1 and induced apoptosis. Transfection of BAECs with LOX-1-specific small interfering RNAs (siLOX-1) minimized baseline LOX-1 production and restrained L5-induced LOX-1 upregulation. Internalization of labeled L1-L5 was monitored in BAECs and human umbilical venous ECs by fluorescence microscopy. LOX-1 knockdown with siLOX-1 impeded the endocytosis of L5 but not L1-L4. In contrast, blocking LDL receptor with RAP (LDL receptor-associated protein) stopped the internalization of L1-L4 but not L5. Although chemically different, L5 and oxLDL competed for EC entry through LOX-1. Via LOX-1, L5 signaling hampered Akt phosphorylation and suppressed EC expression of fibroblast growth factor-2 and Bcl-2. L5 also selectively inhibited Bcl-xL expression and endothelial nitric oxide synthase phosphorylation but increased synthesis of Bax, Bad, and tumor necrosis factor-alpha. Blocking Akt phosphorylation with wortmannin increased LOX-1 expression, suggesting a modulatory role of Akt in LOX-1 synthesis; L5 upregulated LOX-1 by dephosphorylating Akt. Because endothelial nitric oxide synthase and Bcl-2 activities are Akt-dependent, L5 impairs Akt-mediated growth and survival signals in vascular ECs by way of LOX-1. Thus, the L5/LOX-1 complex may play a critical role in atherogenesis and illuminate important targets for disease intervention.

Electronegative LDL circulating in smokers impairs endothelial progenitor cell differentiation by inhibiting Akt phosphorylation via LOX-1.

Endothelial progenitor cells (EPCs), important for endothelial regeneration and vasculogenesis, are reduced by cigarette smoking. To elucidate the mechanisms, we examined the effects of electronegative LDL, circulating in chronic smokers, on EPC differentiation. Using ion-exchange chromatography, we purified smoker LDL into five subfractions, L1-L5. In matched, nonsmoking healthy subjects, L5, the most electronegative subfraction, was either absent or scanty. Sustained L5 treatment inhibited CD31 and KDR expression and EPC differentiation, whereas L1-L4 had no effect. L5 also inhibited telomerase activity to accelerate EPC senescence in correlation with reduced Akt phosphorylation. Transfection of day 3 EPCs with dominant negative Akt constructs inhibited CD31 and KDR expression, stalled EPC differentiation, and promoted early senescence. In contrast, transfection with constitutively active Akt rendered the EPCs resistant to L5, allowing normal maturation. L5 upregulated the lectin-like oxidized low density lipoprotein receptor 1 (LOX-1), and pretreatment of EPCs with TS20, a LOX-1-neutralizing antibody, blocked internalization of L5 by EPCs and prevented L5-mediated inhibition of EPC differentiation. Mixing L5 with L1 to physiological L5/L1 ratios did not attenuate L5's effects. These findings suggest that cigarette smoking is associated with the formation of L5, which inhibits EPC differentiation by impairing Akt phosphorylation via the LOX-1 receptor.

Electronegative LDL impairs vascular endothelial cell integrity in diabetes by disrupting fibroblast growth factor 2 (FGF2) autoregulation.

OBJECTIVE: L5, a circulating electronegative LDL identified in patients with hypercholesterolemia or type 2 diabetes, induces endothelial cell (EC) apoptosis by suppressing fibroblast growth factor (FGF)2 expression. FGF2 plays a pivotal role in endothelial regeneration and compensatory arteriogenesis. It is likely that vasculopathy and poor collateralization in diabetes is a result of FGF2 dysregulation. RESEARCH DESIGN AND METHODS: To investigate this mechanism, we isolated L5 from type 2 diabetic patients. In cultured bovine aortic ECs (BAECs), L5 inhibited FGF2 transcription and induced apoptosis. Because FGF2 stimulates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, we examined whether FGF2 transcription is regulated by Akt through a feedback mechanism. RESULTS: Diabetic L5 reduced FGF2 release to the medium but enhanced caspase-3 activity, with resultant apoptosis. Inhibition of PI3K with wortmannin or suppression of Akt activation with dominant-negative Akt inhibited FGF2 expression. Transfection of BAECs with FGF2 antisense cDNA depleted endogenous FGF2 protein. In these cells, not only was Akt phosphorylation inhibited, but FGF2 transcription was also critically impaired. In contrast, transfecting BAECs with FGF2 sense cDNA augmented Akt phosphorylation. Treatment with constitutively active Akt enhanced FGF2 expression. Augmentation of either FGF2 transcription or Akt phosphorylation rendered BAECs resistant to L5. CONCLUSIONS: These findings suggest that FGF2 is the primary initiator of its own expression, which is autoregulated through a novel FGF2-PI3K-Akt loop. Thus, by disrupting FGF2 autoregulation in vascular ECs, L5 may impair reendothelialization and collateralization in diabetes.

Simvastatin attenuates expression of cytokine-inducible nitric-oxide synthase in embryonic cardiac myoblasts.

Cardiac stem cells or myoblasts are vulnerable to inflammatory stimulation in hearts with infarction or ischemic injury. Widely used for the prevention and treatment of atherosclerotic heart disease, the cholesterol-lowering drugs statins may exert anti-inflammatory effects. In this study, we examined the impact of inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase with simvastatin on the expression of inducible nitric-oxide synthase (iNOS) in embryonic cardiac myoblasts stimulated with the proinflammatory cytokines, interleukin-1 or tumor necrosis factor. Treatment with simvastatin significantly reduced the levels of iNOS mRNA and protein in cytokine-treated rat H9c2 cardiac embryonic myoblasts. Addition of the HMG-CoA reductase product, L-mevalonate, and the by-product of cholesterol synthesis, geranylgeranyl pyrophosphate, could reverse the statin inhibitory effect on iNOS expression. Simvastatin treatment lowered the Rho GTPase activities, whereas the Rho-associated kinase inhibitor Y27632 partially blocked the statin inhibitory effect on nitrite production in the cytokine-treated H9c2 cells. Treatment with simvastatin led to inactivation of NF-kappaB by elevation of the NF-kappaB inhibitor IkappaB and reduction of the NF-kappaB nuclear contents in the cytokine-stimulated H9c2 cells. Hence, treatment with simvastatin can attenuate iNOS expression and NO synthesis in cytokine-stimulated embryonic cardiac myoblasts. The statin inhibitory effect may occur through isoprenoid-mediated intracellular signal transduction, which involves several key signal proteins, such as Rho kinase and IkappaB/NF-kappaB. These data suggest that statin therapy may protect the cardiac myocyte progenitors against the cytotoxicity of cytokine-induced high output of NO production in infarcted or ischemic hearts with inflammation.

[Analysis of platelets in the monitoring of systemic inflammatory response syndrome of critically ill patients].

OBJECTIVE: To study the clinical significance of the variance of platelets in systemic inflammatory response syndrome(SIRS) of critical illness. METHODS: Two hundred and thirteen critically ill patients in ICU, who suffered from SIRS, sepsis and multiple organ dysfunction syndrome (MODS), were enrolled in this study and divided into two groups, survivor group (n=151) and non-survivor group (n=62). Platelet, white blood cell counts and acute physiology and chronic health evaluation II (APACHE II) score were performed immediately after hospitalization, 3 days, 7 days, and 10 days later. At the same time, the serum was collected and the level of tumor necrosis factor-alpha (TNF-alpha) was measured. RESULTS: APACHE II score was much higher, but no difference in the two groups immediately after the hospitalization. However, it increased markedly in non-survivor group, and lowered dramatically in survivor group 7 days after therapy. There was a significant difference between the two groups (P<0.01). Platelets were slightly lower in both groups immediately after the hospitalization. After three days' therapy, it increased to the normal range in the two groups. However, it progressively dropped in non-survivor group 7 days and 10 days later, and it was significantly different from survivor group (P<0.001). The white blood cell counts revealed that there was no significant difference between the two groups. The level of TNF-alpha in serum was much higher in both groups immediately after the hospitalization. After three days' therapy, it further increased and was maintained at the high level in the two groups. However, it progressively dropped in survivor group, while it remained in higher level in non-survivor group 7 days and 10 days later, which was significantly different from survivor group (both P<0.001). CONCLUSION: Refractory thrombocytopenia is sensitively responsive to poor prognosis and severity of SIRS in critical illness.