Sphingomyelin synthase 2 over-expression induces expression of aortic inflammatory biomarkers and decreases circulating EPCs in ApoE KO mice.

AIMS: This study sought to assess the effect of sphingomyelin synthase 2 (SMS2) over-expression on plaque component and endothelial dysfunction in atherosclerosis. MAIN METHODS: We generated recombinant adenovirus vectors containing human SMS2 cDNA (AdV-SMS2) or control gene GFP cDNA (AdV-GFP). Both AdVs were injected (i.v.) into ApoE KO mice to establish SMS2 over-expressing and control mice models, respectively. The mice were fed a high fat diet for 30 days. We then examined their plasma lipid levels, expression levels of aortic inflammatory biomarkers critical for the plaque's stability, and numbers of peripheral endothelial progenitor cells (EPC). KEY FINDINGS: Compared with the control mice, SMS2 over-expression had significantly (1) increased aortic matrix metalloproteinase-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), tissue factor (TF) and cyclooxygenase-2 (COX-2) mRNA levels (1.9-fold, 2.2-fold, 2.6-fold and 3.2-fold, respectively, P<0.01) and protein levels (2.2-fold, 1.9-fold, 1.9-fold and 2.1-fold, respectively, P<0.01); (2) increased MMP-2, COX-2 in situ expression in aortic root (2.6-fold and 2.3-fold, respectively, P<0.01); (3) decreased aortic COX-1 mRNA levels (65%, P<0.01) and protein levels (64%, P<0.01); and (4) decreased CD34/KDR-positive cells (33%, P<0.01), circulating angiogenic cells (CACs) (50%, P<0.05), and colony forming units (CFUs) (40%, P<0.05) in circulation. SIGNIFICANCE: SMS2 over-expression was probably associated with increased expression of aortic inflammatory biomarkers, as well as decreased numbers of CD34/KDR-positive cells, CACs and CFUs in circulation. Therefore, SMS2 over-expression might correlate with endothelial dysfunction and aggravate atherosclerotic plaque instability in ApoE KO mice.

Apolipoprotein A-I inhibits chemotaxis, adhesion, activation of THP-1 cells and improves the plasma HDL inflammatory index.

The anti-inflammatory effects of high density lipoprotein (HDL) are well described, however, such effects of Apolipoprotein A-I (ApoA-I) are less studied. Building on our previous study, we further explored the mechanism of anti-inflammatory effects of ApoA-I, and focused especially on the interaction between monocyte and endothelial cells and plasma HDL inflammatory index in LPS-challenged rabbits. Our results show that ApoA-I significantly decreased LPS-induced MCP-1 release from THP-1 cells and ox-LDL-induced THP-1 migration ratio (P<0.01, respectively). ApoA-I significantly decreased sL-selectin, sICAM-1 and sVCAM-1 release (P<0.01, P<0.01, P<0.05, respectively) from LPS-stimulated THP-1 cells. Furthermore, ApoA-I significantly inhibited LPS-induced CD11b and VCAM-1 expression on THP-1 cells (P<0.01, P<0.05, respectively). ApoA-I diminished LPS-induced mCD14 expression (P<0.01) and NFkappaB nuclear translocation in THP-1 cells. After single dose treatment of ApoA-I, the value of plasma HDL inflammatory index in LPS-challenged rabbits was improved significantly (P<0.05). These results suggest that ApoA-I can inhibit chemotaxis, adhesion and activation of human monocytes and improve plasma HDL inflammatory index with presenting beneficial anti-inflammatory effects.

Human ApoA-I overexpression diminishes LPS-induced systemic inflammation and multiple organ damage in mice.

Apolipoprotein A-I (ApoA-I) is the major apolipoprotein of high density lipoprotein (HDL). To investigate the protective effect of ApoA-I against lipopolysaccharide (LPS)-induced systemic inflammation and multiple organ damage in mice, we established a human ApoA-I overexpression mouse model using recombinant adenovirus vector (AdV-AI). The histomorphologic analysis showed that AdV-AI administration greatly attenuated LPS-induced acute injury in lung and kidney. AdV-AI treatment also significantly inhibited LPS-induced increments of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta levels in serum (P < 0.01, P < 0.05 and P < 0.05, respectively) and in bronchoalverolar lavage fluid (P < 0.05, respectively), and of serum creatine kinase and creatinine levels (P < 0.05, respectively). Moreover, we found that the increments of CD14 expression in liver and lung induced by LPS were significantly reduced by AdV-AI treatment (P < 0.05 and P < 0.01, respectively). In conclusion, adenovirus-mediated ApoA-I overexpression plays a protective effect against LPS-induced systemic inflammation and multiple organ damage in mice. Such effect may attribute partly to the suppression of inflammatory cytokine release and reduction of CD14 expression.

Apolipoprotein A-I attenuates renal ischemia/reperfusion injury in rats.

Apolipoprotein A-I (ApoA-I), the major protein component of serum high-density lipoprotein (HDL), exhibits its anti-inflammatory activity in inflammatory responses. As renal inflammation plays an important role in ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the beneficial effect of ApoA-I on renal I/R injury in rats and the underlined mechanism. Using rats subjected to renal I/R by occlusion of bilateral renal pedicles, we found that administration of ApoA-I significantly reduced serum creatinine levels, serum TNF-alpha and IL-1beta levels as well as tissue myeloperoxidase (MPO) activity, compared with I/R controls. Moreover, ApoA-I treatment suppresses the expression of intercellular adhesion molecules-1 (ICAM-1) and P-selectin on endothelium, thus diminishing neutrophil adherence and the subsequent tissue injury. These results showed that ApoA-I reduced I/R-induced inflammatory responses, decreased renal microscopic damage and improved renal function. It seems likely that ApoA-I protects kidney from I/R injury by inhibiting inflammatory cytokines release and neutrophil infiltration and activation.

Apolipoprotein A-I diminishes acute lung injury and sepsis in mice induced by lipoteichoic acid.

Lipoteichoic acid (LTA), as a primary immunostimulus, triggers the systematic inflammatory responses. Our hypothesis is that ApoA-I can neutralize LTA toxicity, like its effect on LPS. BALB/c mice were challenged with LTA, followed by human ApoA-I administration. We found that ApoA-I could attenuate LTA-induced acute lung injury and inflammation and significantly inhibit LTA-induced IL-1beta and TNF-alpha accumulation in the serum (P<0.01 and P<0.05, respectively), as well as in bronchoalveolar lavage (BAL) fluid (P<0.01 and P<0.05, respectively). Moreover, ApoA-I could significantly reduce the L-929 cell mortality caused by LTA-activated macrophages in a dose-dependent fashion. Furthermore, ApoA-I treatment could diminish LTA-mediated NFkappaB nuclear translocation in macrophages. An in vitro binding assay indicated that ApoA-I can bind LTA. These results clearly indicated that ApoA-I can effectively protect against LTA-induced sepsis and acute lung damage. The mechanism might be related to the binding and neutralization of LTA.

SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis.

Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergent-insoluble microdomains and significantly increased tumor necrosis factor-alpha-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.

The protective effect of ApolipoproteinA-I on myocardial ischemia-reperfusion injury in rats.

It is well established that reperfusion of heart is the optimal method for salvaging ischemic myocardium, however, the success of this therapy could be limited by reperfusion injury, which is involved in inflammatory responses. High density lipoprotein (HDL) has an anti-inflammatory function and can protect the heart from ischemia-reperfusion (I/R) injury. In this study, we investigated the cardioprotective role of apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, in I/R injury. Using rats subjected to myocardial I/R by ligation of left anterior descending coronary artery (LAD), we found that administration of ApoA-I (20 mg/kg, iv) before the onset of reperfusion of myocardial infarction can significantly reduce serum creatine kinase (CK) levels (62.1+/-13.8%, p<0.01) and heart TNF-alpha as well as IL-6 levels, compared with saline controls (40.4+/-14.7%, 44+/-9.8%, p<0.01 respectively). Moreover, ApoA-I treatment suppresses the expression of ICAM-1 on endothelium, thus diminishing neutrophil adherence, transendothelial migration, and the subsequent myocyte injury. We concluded that ApoA-I could effectively protect rat heart from I/R injury.

Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice.

High density lipoprotein (HDL) binds lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of our study was to investigate the effects of Apolipoprotein (ApoA-I), the major apolipoprotein of HDL, on LPS-induced acute lung injury (ALI) and endotoxemia. BALB/c mice were challenged with LPS, followed by ApoA-I or saline administration for 24h. The mice were then sacrificed and histopathological analysis of the lung was performed. We found that ApoA-I could attenuate LPS-induced acute lung injury and inflammation. To investigate the mechanisms, we measured tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels in the serum and bronchoalveolar lavage (BAL) fluid and found that ApoA-I could significantly inhibit LPS-induced increases in the IL-1beta and TNF-alpha levels in serum (P<0.05, respectively), as well as in the IL-1beta, TNF-alpha, and IL-6 levels in BAL fluid (P<0.01 and P<0.05, P<0.05, respectively). Moreover, we evaluated the effect of ApoA-I on the mortality of L-929 cells which were attacked by LPS-activated peritoneal macrophages. We found that ApoA-I could significantly inhibit the LPS-induced cell death in a dose-dependent fashion. Furthermore, we investigated in vivo the effects of ApoA-I on the mortality rate and survival time after LPS administration and found that ApoA-I significantly decreased the mortality (P<0.05) and increased the survival time (P<0.05). In summary, the results suggest that ApoA-I could effectively protect against LPS-induced endotoxemia and acute lung damage. The mechanism might be related to inhibition of inflammatory cytokine release from macrophages.

Neutrophils activation can be diminished by apolipoprotein A-I.

High density lipoprotein (HDL) has anti-inflammatory function. To investigate the effects of apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, on activated neutrophils, we stimulated neutrophils in vitro with fMLP and PMA, as a receptor-binding and a nonreceptor-binding stimuli, respectively, and incubated ApoA-I with those neutrophils. Three conditions were utilized: 1) resting neutrophils + ApoA-I (0, 2.5,5, 10 microg/mL respectively), 2) fMLP(10(-7) mol/L)-activated neutrophils + ApoA-I (0, 2.5, 5, 10 microg/mL respectively), and 3) PMA(10(-7) mol/L)-activated neutrophils + ApoA-I (0, 2.5, 5, 10 microg/mL respectively). After incubation, we measured neutrophils adhesion to fibronectin, oxidative bust (O2- and H2O2 production), degranulation (release of MPO and elastase), and L929 cell mortality which were attacked by release-out of cytokines in activated neutrophils (using MTT). Our results showed that in vitro ApoA-I inhibits fMLP- and PMA- activated neutrophil adhesion, oxidative burst, degranulation and L929 cell mortality. These inhibition effects of ApoA-I on fMLP-activated neutrophils are more powerful than that on PMA-activated neutrophils. ApoA-I has no effect on resting neutrophils. We concluded that ApoA-I could diminish the function of activated neutrophils.

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells.

AIM: To investigate the possibility of recombinant high-density lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26+/-5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 microg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 microg/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs 3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 microg/mL. Cytotoxicity of the rHDL-ACM to SMMC-7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5 microg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes. HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.

Role of apolipoprotein A-I in protecting against endotoxin toxicity.

High density lipoprotein (HDL) binds lipopolysaccharide (LPS or endotoxin) and neutralizes its toxicity. We investigated the function of Apolipoprotein A-I (ApoA-I), a major apolipoprotein in HDL, in this process. Mouse macrophages were incubated with LPS, LPS+ApoA-I, LPS+ApoA-I+LFF (lipoprotein-free plasma fraction d>1.210 g/ml), LPS+HDL, LPS+HDL+LFF, respectively. MTT method was used to detect the mortality of L-929 cells which were attacked by the release-out cytokines in LPS-activated macrophages. It was found that ApoA-I significantly decreased L-929 cells mortality caused by LPS treatment (LPS vs. LPS+ApoA-I, P<0.05) and this effect became even more significant when LFF was utilized (LPS vs. LPS+ApoA-I+LFF, P<0.01; LPS vs. LPS+HDL+LFF, P<0.01). There was no significant difference between LPS+ApoA-I+LFF and LPS+HDL+LFF treatment, indicating that ApoA-I was the main factor. We also investigated in vivo effects of ApoA-I on mouse mortality rate and survival time after LPS administration. We found that the mortality in LPS+ApoA-I group (20%) and in LPS+ApoA-I+LFF group (10%) was significantly lower than that in LPS group (80%) (P<0.05, P<0.01, respectively); the survival time was (43.20 +/- 10.13) h in LPS+ApoA-I group and (46.80 +/- 3.79) h in LPS+ApoA-I+LFF group, which were significantly longer than that in LPS group (16.25 +/- 17.28) h (P<0.01). We also carried out in vitro binding study to investigate the binding capacity of ApoA-I and ApoA-I+LFF to fluorescence labeled LPS (FITC-LPS). It was shown that both ApoA-I and ApoA-I+LFF could bind with FITC-LPS, however, the binding capacity of ApoA-I+LFF to FITC-LPS (64.47 +/- 8.06) was significantly higher than that of ApoA-I alone (24.35 +/- 3.70) (P<0.01). The results suggest that: (1) ApoA-I has the ability to bind with and protect against LPS; (2) LFF enhances the effect of ApoA-I; (3) ApoA-I is the major contributor for HDL anti-endotoxin function.

Metabolism of Oxidized High Density Lipoprotein-2(ox-HDL(2)) in Rat Hepatic Sinusoidal Cells.

Incubation of rat hepatic sinusoidal cells with FITC-HDL(2), FITC-ox-HDL(2) and [(3)H]CE-HDL(2)(rHDL(2)), ox-rHDL(2) showed that binding of FITC-HDL(2) to the cells was competitive to ox-HDL(2), but not to HDL(2). The cell-endocytic fluorescence strength (FS) of FITC-HDL(2) and radioactivity of ox-rHDL(2) were 45.5% of that of FITC-HDL(2) and 61.4% of that of rHDL(2), respectively. Endocytic FS and radioactivity were mainly in TCA-precipitable and supernatant part, respectively. The cell-released FS and radioactivity were 67.7% and 10.9% of the cell-endocytic FS and the radioactivity, respectively, and both of them were mainly TCA-precipitable. These results suggest that: (1) There is probably an ox-HDL receptor on the surface of rat hepatic sinusoidal cells, which is different from HDL receptor. (2) The metabolic behaviour of ox-rHDL(2) in the cells is similar to HDL(2). Both of them do not take a lysosomal pathway. Apoproteins and CE components dissociate from endocytic lipoprotein in the cells. After the cells have taken up most of CE, the residual CE recombines with apolipoprotein to form a lipoprotein and is released from the cells by retroendocytosis. (3) Oxidative modification of HDL(2) weakens its ability to cholesterol reverse transport.

Cooperation of HDL Receptor and Hepatic Lipase in the Selective Uptake of HDL(2)-CE by Rat Hepatic Sinusoidal Cells.

The recombined (3)H-CE-HDL(2)(rHDL(2)) keeps the biological activities of the native HDL(2). After rat hepatic sinusoidal cells were incubated with rHDL(2) at 37 degrees for 3 h (normal group), the cell-endocytic cpm was 995-/+147(mean-/+s, n=2). After the cells were further incubated for 2 h, the cell-release TCA-precipitable cpm and the TCA-supernatant cpm were 78-/+32 and 12-/+9 respectively. These values were 339-/+62, 19-/+11 and 9-/+5 respectively in the acetylimidazole-modified group, and 542-/+78, 34-/+14 and 9-/+8 respectively in the heparin-pretreated group. Our results suggested that: (1) Rat hepatic sinusoidal cells internalize HDL(2) and take up HDL(2)-CE by its HDL receptor, and HDL(3) was secreted out of the cells by retroendocytosis. (2) Hepatic lipase (HL) induces directly the selective uptake of HDL(2)-CE by the cells. (3) There is cooperation between the HDL receptor and HL in the selective uptake of HDL(2)-CE by the cells.