Cholesterol efflux capacity.

<p>Apolipoprotein B (apoB)-depleted sera of healthy subjects (control, n = 27) and patients with age-related macular degeneration (AMD, n = 29) were examined for (A) their ability to promote [³H]-cholesterol efflux from macrophages. [³H]-cholesterol-labeled RAW264.7 macrophages were incubated with 2.8% apoB-depleted sera for 4 hours. Cholesterol efflux is expressed as radioactivity in the supernatant relative to total radioactivity (in supernatant and cells). Values shown represent means of two independent experiments.</p

Laropiprant antagonizes the increased platelet aggregation by TP and EP3 receptor activation

<p>In (<b>A</b>), aggregation was induced by U46619 (300 nmol/L) which was concentration-dependently inhibited by laropiprant (n = 4). In <b>B–G</b>, ADP concentrations (1.25–10 μmol/L) were adjusted to give 30–50% of maximal aggregation. (<b>B</b>) The inhibiton of ADP-induced aggregation by the EP4 agonist CAY10598 (300 nmol/L) and the IP agonist iloprost (3 nmol/L) was not affected by laropiprant (10 nmol/L, (n = 7)). (<b>C</b>) The EP3 agonist sulprostone concentration dependently amplified ADP-induced aggregation (n = 4–6). (<b>D</b>) The effect of sulprostone (300 nmol/L) was concentration dependently inhibited by laropiprant (n = 4). (<b>E</b>) Pretreatment with acetylsalicylic acid (1 mmol/L) markedly attenuated the pro-aggregatory effect of sulprostone, and in this case, laropiprant was unable to reverse the stimulatory effect of the EP3 agonist. Data were expressed as percent of control response. (<b>F</b>) The TP receptor antagonist SQ29578 (1 µmol/L) inhibited the sulprostone-induced increase in platelet aggregation to the same extend as laropiprant (10 µmol/L). The combination of SQ29578 and laropiprant did not cause further inhibition as compared to laropiprant or SQ29578 alone (n = 4–6) (<b>G</b>) The pro-aggregatory effect of sulprostone was not inhibited by the EP1 receptor antagonist SC51322 (n = 4). Data were expressed as percent of control ADP response and are shown as mean+SEM. *P<0.05 as compared to vehicle and^#P<0.05 as compared to the respective agonist treatment.</p

Laropiprant antagonizes Ca²⁺ mobilization induced by TP receptor activation.

<p>Ca²⁺ responses were detected by flow cytometry as changes in fluorescence of the Ca²⁺ -sensitive dye Fluo-3 by flow cytometry and are presented as percent of baseline fluorescence. (<b>A</b>) The TP agonist, U46619 (3–3000 nmol/L), induced Ca²⁺flux in a concentration-dependent manner and this effect was completely inhibited by laropiprant at 10 μmol/L (n = 6). (<b>B</b>) The EP4 receptor agonist CAY10598 (300 nmol/L) and (<b>C</b>) the IP receptor agonist iloprost (3 nmol/L) caused a significant inhibition of the ADP-induced Ca²⁺ flux (n = 4). Laropiprant (10 µmol/L) did not antagonize these effects (n = 4). Values are shown as mean+SEM. *P<0.05 as compared to vehicle and ^#P<0.05 as compared to the respective agonist treatment treatment.</p

DP receptor activation inhibits platelet aggregation and intracellular Ca²⁺ mobilization, effects that are counteracted by laropiprant.

<p>(<b>A–E</b>) Aggregation was induced using ADP or collagen at concentrations which were adjusted to give submaximal aggregation. Data were expressed as percent of the control response. (<b>A</b>) ADP-induced aggregation was inhibited by PGD₂ (30 nmol/L) and this effect was concentration dependently counteracted by laropiprant (n = 6). (<b>B</b>) The ADP- induced aggregation was slightly reduced by the DP receptor antagonist BWA868c (1 μmol/L), but not by laropiprant (1 μmol/L) alone. PGD₂ (30 nmol/L) and the DP agonist, BW245c (3 nmol/L) caused pronounced inhibition of platelet aggregation and these effects were reversed by BWA868c and laropiprant (n = 4). (<b>C</b>) Pre-treatment of platelets with acetylsalicylic acid (1 mmol/L) did not affect the inhibitory effects of PGD₂ (30 nmol/L) and BW245c (3 nmol/L) and the reversal of these effects by BWA868c and laropiprant at 1 μmol/L (n = 4). (<b>D</b>) Laropiprant caused a concentration-dependent inhibition of collagen-induced aggregation (n = 6). (<b>E</b>) Laropiprant (1 μmol/L) counteracted the inhibition of aggregation by PGD₂ (30 nmol/L) and BW245c (3 nmol/L) (n = 4–6). (<b>F, G</b>) Ca²⁺ responses were detected by flow cytometry as changes in fluorescence of the Ca²⁺-sensitive dye Fluo-3 by and are presented as percent of baseline fluorescence. Ca²⁺ flux induced by ADP (30–1000 nmol/L) was significantly reduced by pre-treatment of platelets with (<b>E</b>) PGD₂ (30 nmol/L) and (<b>F</b>) BW245c (3 nmol/L). The inhibition of the Ca²⁺ flux by DP receptor activation was reversed by laropiprant (1 μmol/L) and the DP antagonist, BWA868c (1 μmol/L) (n = 6). Values are shown as mean+SEM. *P<0.05 as compared to vehicle and # P<0.05 as compared to agonist treatment.</p

Pearson correlation of clinical and laboratory variables with HDL AE activity and cholesterol efflux capacity.

<p>Pearson correlation of clinical and laboratory variables with HDL AE activity and cholesterol efflux capacity.</p

Baseline characteristics, biochemical laboratory parameters, comorbidities, and classification of AHF patients (n = 152).

<p>Baseline characteristics, biochemical laboratory parameters, comorbidities, and classification of AHF patients (n = 152).</p

Paraoxonase activity.

<p>Activity of HDL-associated paraoxonase was measured using phenylacetate as substrate. Paraoxonase activity of apoB-depleted sera was calculated from the slopes of the kinetic chart. Values shown represent means of four independent experiments.</p

Univariable logistic regression analysis of HDL function, clinical and laboratory parameters and hospital mortality.

<p>Univariable logistic regression analysis of HDL function, clinical and laboratory parameters and hospital mortality.</p

HDL-apolipoproteins and HDL associated lipids.

<p>Levels of total cholesterol (A), non-esterified cholesterol (FC) (B), phospholipids (PL) (C), free fatty acids (FFA) (D), triglycerides (TG) (E) were measured enzymatically in apoB depleted serum. HDL associated apolipoproteins ApoA-I (F), apoA-II (G), apoC-II (H), apoC-III (I) and apoE (J) were determined in apoB-depleted serum by immunoturbidimetry.</p

Anti-inflammatory capacity.

<p>(A) U937 monocytes containing a reporter cassette for factor-κB (NF-κB) were pretreated in the absence and presence of 10% lipoprotein deficient sera (LPDS) in the presence of indicated concentrations of reconstituted HDL (rHDL). After 1 ½ hours, cells were stimulated with LPS (50 ng/ml) for 24 hours, followed by assessment of GFP expression by flow cytometry. (B) ApoB-depleted sera of healthy subjects and AMD patients were analyzed for their ability to inhibit lipopolysaccharide LPS-induced NF-κB activation in monocytes. U937 monocytes were pretreated with 7% apoB- depleted sera. After 1 ½ hours, cells were stimulated with LPS (50 ng/ml) for 24 hours, followed by assessment of GFP expression by means of flow cytometry. Values shown represent means of two independent experiments.</p