Oxidized Phospholipids on Lipoprotein(a) Elicit Arterial Wall Inflammation and an Inflammatory Monocyte Response in Humans

Abstract

Background: Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent cardiovascular risk factor, but the underlying mechanisms responsible for its pathogenicity are poorly defined. Because Lp(a) is the prominent carrier of proinflammatory oxidized phospholipids (OxPLs), part of its atherothrombosis might be mediated through this pathway.

Methods: In vivo imaging techniques including magnetic resonance imaging, (18)F-fluorodeoxyglucose uptake positron emission tomography/computed tomography and single-photon emission computed tomography/computed tomography were used to measure subsequently atherosclerotic burden, arterial wall inflammation, and monocyte trafficking to the arterial wall. Ex vivo analysis of monocytes was performed with fluorescence-activated cell sorter analysis, inflammatory stimulation assays, and transendothelial migration assays. In vitro studies of the pathophysiology of Lp(a) on monocytes were performed with an in vitro model for trained immunity.

Results: We show that subjects with elevated Lp(a) (108 mg/dL [50-195 mg/dL]; n=30) have increased arterial inflammation and enhanced peripheral blood mononuclear cells trafficking to the arterial wall compared with subjects with normal Lp(a) (7 mg/dL [2-28 mg/dL]; n=30). In addition, monocytes isolated from subjects with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation (n=15). In vitro studies show that Lp(a) contains OxPL and augments the proinflammatory response in monocytes derived from healthy control subjects (n=6). This effect was markedly attenuated by inactivating OxPL on Lp(a) or removing OxPL on apolipoprotein(a).

Conclusions: These findings demonstrate that Lp(a) induces monocyte trafficking to the arterial wall and mediates proinflammatory responses through its OxPL content. These findings provide a novel mechanism by which Lp(a) mediates cardiovascular disease.

Clinical trial registration: URL: http://www.trialregister.nl. Unique identifier: NTR5006 (VIPER Study).

Keywords: atherosclerosis; lipoproteins; monocytes.

Figure 1. Increased arterial wall inflammation in subjects with elevated Lp(a)

(A) Cross-sectional ¹⁸F-FDG PET/CT images demonstrating an increased ¹⁸F-FDG uptake (yellow) in the left carotid (top, indicated by white arrow) and aorta (bottom) in a subject with normal Lp(a) (left) and a subject with elevated Lp(a) (right), quantified as the maximum target to background ratio (TBR) in the (B) carotid arteries, and (C) ascending aorta in subjects with elevated Lp(a) (n=30) and normal Lp(a) (n=30), (D) cross-sectional SPECT/CT images demonstrating increased autologous ^99mTc-labeled PBMCs accumulation (blue; at T=6 hours post-infusion), depicted as the arterial wall to blood pool ratio (ABR) at the level of (E) the carotids and (F) ascending aorta in subjects with elevated Lp(a) (n=15) and normal Lp(a) (n=15). **=p<0.01.

Figure 2. Monocytes have an activated and inflammatory phenotype in Lp(a) subjects

(A) Bar graphs display the expression (quantified as delta MFI) of chemokine, adhesion and transmigration markers on monocytes as assessed with flow cytometry in subjects with elevated Lp(a) (n=15, black bars) compared with normal Lp(a) (n=15, grey bars), (B) bar graph and (C) microscope images demonstrating increased endothelial transmigration of monocytes (calculated per at least 4 fields of view) isolated from subjects with elevated Lp(a) (n=15, black bar) compared with normal Lp(a) (n=15, grey bar), coinciding with augmented spreading and adhesion as illustrated nucleus (Hoechst, blue) and F-actin (green) stain, (D) bar graphs showing expression (quantified as delta MFI) of scavenger and other receptors on monocytes in subjects with elevated Lp(a) (n=15, black bars) compared with subjects with normal Lp(a) (n=15, grey bars), (E-H) in response to an overnight challenge to Pam3Cys (10 μg/ml), monocytes isolated from subjects with elevated Lp(a) (n=15, black bars) produced higher levels of IL-1β (E), IL-6 (F) and TNFα (G) and lower levels of IL-10 (H), compared with monocytes of subjects with normal Lp(a) (n=15, grey bars). ^=p<0.06, *=p<0.05, **=p<0.01, ***=p<0.001.

Figure 3. Isolated Lp(a) primes monocytes towards a responsive state

(A,B) priming of healthy monocytes with plasma of a subject with high Lp(a) (1^st grey bar) for 24h resulted in an increased production of TNFα and IL-6 upon re-stimulation with Pam3Cys (10 μg/mL) on day 6, compared with plasma of normal Lp(a) (1^st white bar). In addition, after lipid-depletion (2^nd white and grey bar) this effect of high Lp(a) plasma was profoundly reduced compared to the undepleted control (n=6), (C,D) priming of healthy monocytes with β-glucan (5 μg/mL, positive control, black bar) or Lp(a) (grey bar, 250 μg/mL) for 24h, induced an increased production of TNFα and IL-6 upon re-stimulation with Pam3Cys (10 μg/mL) on day 6, compared with priming with RPMI (negative control, white bar). LDL (grey bar, 10 μg/mL) or HDL (grey bar, 10 μg/mL) did not induce this increase (n=6) (E,F) in addition, various concentrations of Lp(a) (grey bars) for 24h, resulted in a dose-dependent increased production of TNFα and IL-6 upon re-stimulation with Pam3Cys (10 μg/mL) on day 6 (n=6) compared to priming with RPMI (negative control, white bar) (G,H) priming of healthy monocytes with LDL (10 μg/mL) (grey bar) did not result in higher cytokine levels after Pam3Cys (10 μg/mL) compared to the negative control RPMI (white bar), in contrast to β-glucan (5 μg/mL, positive control, black bar) (n=6), whereas (I,J) priming of healthy monocytes with β-glucan (5 μg/mL, positive control, black bar) or the r-apo(a) construct 8K-IV (grey bars) induced increased cytokine levels after Pam3Cys (10 μg/mL) (n=6), compared with RPMI (negative control, white bar). *=p<0.05, **=p<0.01, ***=p<0.001.

Figure 4. OxPL induce the enhanced monocyte response

(A) Immunoblotting of purified apo(a), Lp(a) and LDL; OxPL, apo(a), and apoB-100 resolved on SDS-PAGE in reducing and non-reducing conditions, were detected using the monoclonal antibodies E06, LPA4 and MB47 respectively. E06 immunostained apo(a) and Lp(a) but not LDL isolated from the same subject, (B,C) pre-treatment with the E06 antibody (1 nM) against OxPL inhibited the increased monocyte response after priming with 8K-IV (0.1 μg/mL, grey bars) and subsequent challenge with Pam3cys, shown as % of the initial priming with 8K-IV, corrected for IgM control antibody (n=6), (D,E) priming with β-glucan (5 μg/mL, positive control, black bar) and the r-apo(a) construct 17K (0.1 μg/mL, grey bar) induced increased cytokine production after Pam3Cys (10 μg/mL), whereas 17KΔLBS lacking OxPL (0.1 μg/mL, grey bar) did not (n=6), (F,G) priming with β-glucan (5 μg/mL positive control, black bar) and oxPAPC (grey bars) increased the monocyte responsiveness compared with RPMI (negative control, white bar) (n=6). ^=p<0.06, *=p<0.05, **=p<0.01, ***=p<0.001.