High-Density Lipoprotein Function in Exudative Age-Related Macular Degeneration.

PURPOSE:High-density lipoproteins (HDL) have long been implicated in the pathogenesis of age-related macular degeneration (AMD). However, conflicting results have been reported with regard to the associations of AMD with HDL-cholesterol levels. The present study is the first to assess HDL composition and metrics of HDL function in patients with exudative AMD and control patients. METHODS:Blood samples were collected from 29 patients with exudative AMD and 26 age-matched control patients. Major HDL associated apolipoproteins were determined in apoB-depleted serum by immunoturbidimetry or ELISA, HDL-associated lipids were quantified enzymatically. To get an integrated measure of HDL quantity and quality, we assessed several metrics of HDL function, including cholesterol efflux capacity, anti-oxidative and anti-inflammatory activities using apoB-depleted serum from study participants. RESULTS:In our study, we observed that the HDL associated acute phase protein serum amyloid A (SAA) was significantly increased in AMD patients (p<0.01), whereas all other assessed apolipoproteins including ApoA-I, apoA-II, apoC-II, apoC-III and apoE as well as major HDL associated lipids were not altered. HDL efflux capacity, anti-oxidative capacity and arylesterase activity were not different in AMD patients when compared with the control group. The ability of apoB-depleted serum to inhibit monocyte NF-κB expression was significantly improved in AMD patients (mean difference (MD) -5.6, p<0.01). Moreover, lipoprotein-associated phospholipase A2 activity, a marker of vascular inflammation, was decreased in AMD subjects (MD -24.1, p<0.01). CONCLUSIONS:The investigated metrics of HDL composition and HDL function were not associated with exudative AMD in this study, despite an increased content of HDL associated SAA in AMD patients. Unexpectedly, anti-inflammatory activity of apoB-depleted serum was even increased in our study. Our data suggest that the investigated parameters of serum HDL function showed no significant association with exudative AMD. However, we cannot exclude that alterations in locally produced HDL may be part of the AMD pathogenesis

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

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

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

Anti-oxidative capability.

<p>The anti-oxidative activity of HDL was determined by inhibition of AAPH-initiated oxidation of the fluorescent dye dihydrorhodamine (DHR). Incubation of DHR in presence of apoB-depleted sera from healthy subjects or AMD patients led to a reduction in the oxidation of DHR. Values shown represent means of two independent experiments.</p

Lipoprotein associated phospholipase A2 (Lp-PLA2) activity.

<p>Lipoprotein associated phospholipase A2 (Lp-PLA2) activity of apoB-depleted sera was measured using 2-thio PAF as substrate. Lp-PLA2 activity of apoB-depleted sera was calculated from the slopes of the kinetic chart. Values shown represent means of two independent experiments.</p