Lipoprotein(a) and Oxidized Phospholipids Promote Valve Calcification in Patients With Aortic Stenosis

Background Lipoprotein(a) [Lp(a)], a major carrier of oxidized phospholipids (OxPL), is associated with an increased incidence of aortic stenosis (AS). However, it remains unclear whether elevated Lp(a) and OxPL drive disease progression and are therefore targets for therapeutic intervention. Objectives This study investigated whether Lp(a) and OxPL on apolipoprotein B-100 (OxPL-apoB) levels are associated with disease activity, disease progression, and clinical events in AS patients, along with the mechanisms underlying any associations. Methods This study combined 2 prospective cohorts and measured Lp(a) and OxPL-apoB levels in patients with AS (Vmax >2.0 m/s), who underwent baseline 18F-sodium fluoride (18F-NaF) positron emission tomography (PET), repeat computed tomography calcium scoring, and repeat echocardiography. In vitro studies investigated the effects of Lp(a) and OxPL on valvular interstitial cells. Results Overall, 145 patients were studied (68% men; age 70.3 ± 9.9 years). On baseline positron emission tomography, patients in the top Lp(a) tertile had increased valve calcification activity compared with those in lower tertiles (n = 79; 18F-NaF tissue-to-background ratio of the most diseased segment: 2.16 vs. 1.97; p = 0.043). During follow-up, patients in the top Lp(a) tertile had increased progression of valvular computed tomography calcium score (n = 51; 309 AU/year [interquartile range: 142 to 483 AU/year] vs. 93 AU/year [interquartile range: 56 to 296 AU/year; p = 0.015), faster hemodynamic progression on echocardiography (n = 129; 0.23 ± 0.20 m/s/year vs. 0.14 ± 0.20 m/s/year] p = 0.019), and increased risk for aortic valve replacement and death (n = 145; hazard ratio: 1.87; 95% CI: 1.13 to 3.08; p = 0.014), compared with lower tertiles. Similar results were noted with OxPL-apoB. In vitro, Lp(a) induced osteogenic differentiation of valvular interstitial cells, mediated by OxPL and inhibited with the E06 monoclonal antibody against OxPL. Conclusions In patients with AS, Lp(a) and OxPL drive valve calcification and disease progression. These findings suggest lowering Lp(a) or inactivating OxPL may slow AS progression and provide a rationale for clinical trials to test this hypothesis.

A ortic valve stenosis (AS) is the commonest form of valvular heart disease in developed countries, and its disease burden is anticipated to double over the next 50 years (1). Following the failure of statins in reducing AS progression (2), effective medical therapies are lacking. The only treatment option is surgical or transcatheter aortic valve replacement (AVR), after patients develop severe stenosis and symptoms (3). Patients with AS are elderly, often have multiple comorbidities and are not well suited to major surgery or intervention. Moreover, these procedures are expensive and are associated with perioperative as well as long-term morbidity and mortality. There is, therefore, an unmet clinical need for novel therapies to slow AS progression, thereby obviating the requirement for AVR altogether.
Lipoprotein(a) (Lp[a]) is a major carrier of oxidized phospholipids (OxPL) (4,5) and has been established as a causal risk factor for AS in several genetic and population studies (6)(7)(8). Lp(a) is unaffected by statin therapy, but levels can now be reduced with novel compounds (9), making Lp(a) a potential therapeutic target in AS. The risk factors associated with incident AS may differ from those associated with disease progression, due to pathophysiological differences in the initiation and propagation phases of the disease (10). Before Lp(a) or its associated OxPL can be considered viable targets for therapeutic intervention in AS, prospective longitudinal studies are required to establish whether Lp(a) and OxPL are associated with disease progression-in addition to mechanistic work investigating the cellular mechanisms linking Lp(a) and OxPL with valvular calcification.
In a prior study investigating patients with mild to moderate AS, elevated Lp(a) and OxPL on apolipoprotein B-100 (apoB) were associated with higher rates of disease progression determined by echocardiography, as well as an increased need for AVR (11). However, this finding has not been validated in In this study, we analyzed a clinically representative cohort of patients with AS and compared baseline Lp(a) and OxPL-apoB levels to calcification activity in the aortic valve using 18

METHODS
STUDY POPULATIONS. Patients investigated in this study were drawn from across 2 multimodality imaging studies that prospectively collected data with respect to AS progression and adverse clinical events: the Ring of Fire study (12)  In the Ring of Fire study, patients age >50 years and matched control subjects were recruited from the outpatient department of the Edinburgh Heart Centre. PET, CT calcium scoring, and echocardiography were performed to assess disease activity and progression in a total of 81 patients.  Table 1).

LP(A), OxPL AND AUTOTAXIN MEASUREMENTS.
Plasma Lp(a) and OxPL-apoB were measured in all patients in both cohorts using chemiluminescent immunoassays, as previously described (11). Autotaxin (ATX) associated with Lp(a) and apoB was also measured (5).

BASELINE CALCIFICATION ACTIVITY WITH 18 F-NaF
PET. 18 F-NaF PET provides a measure of valvular calcification activity in aortic stenosis, binding preferentially to developing tissue microcalcification and predicting AS progression (12,(14)(15)(16)(17). In this study, aortic valve calcification activity was assessed at baseline in the Ring of Fire cohort using 18  Zheng et al.     Zheng et al.   Table 2

STUDY COHORT AND BASELINE CHARACTERISTICS.
After combining available data from the Ring of Fire cohort and the SALTIRE trial, a total of 145 elderly patients had samples available for Lp(a) and OxPL-apoB measurements (Figures 2A and 2B). In both studies, disease severity assessed by echocardiography was comparable (Online Table 3 Whether this increased calcification activity translated into faster progression of aortic valve calcium burden was subsequently assessed using repeat CT  Table 5).    Zheng et al.  p ¼ 0.054) (Online Table 5).  Figures 1C and D).  (Figures 5D to 5F). In addition, when assessing cell morphology, 17K-WT induced an activated rhomboid shape, suggesting VIC activation or phenotype transformation. In contrast, VICs exposed to 17KDLBS10 demonstrated a spindleshaped morphology, corresponding to a quiescent state ( Figures 5G and 5H).

DISCUSSION
In this multimodality imaging study, we present the novel finding that increased Lp(a) and OxPL-apoB levels in elderly patients with advanced AS are associated with increased valvular calcification activity using 18 F-NaF PET and confirmed faster rates of disease progression using both CT calcium scoring and echocardiography (Central Illustration). This translated into an increased incidence of AVR and death.
In vitro studies demonstrated that these observations