Synergetic Effect of Lipoprotein(a) and Lipoprotein-Associated Phospholipase A2 on Functional Outcomes in Patients with Ischemic Stroke

The relationship of lipoprotein(a) [Lp(a)] and stroke functional outcomes was conicting. The relationship of Lp(a) and Lp-PLA 2 levels to functional outcomes is unclear. The aim was to clarify whether high Lp(a) is associated with poor functional outcomes and examine the relationship of Lp(a) and Lp-PLA 2 to functional outcomes in patients with ischemic stroke. A total of 10,422 individuals from the third China National Stroke Registry cohort were recruited. Plasma level of Lp(a) at admission was measured with enzyme-linked immunosorbent assay. The cut-off was set at the median for Lp(a). Functional outcome was assessed using the modied Rankin scale (mRS) at 3 months after stroke. The association between Lp(a) and stroke functional outcomes was evaluated using a multivariate Cox regression model. a synergetic with Lp-PLA on functional outcomes after ischemic the between Lp(a) and poor in with

In ammation is involved in the occurrence and development of poor functional outcomes in patients with ischemic stroke. 7 Lipoprotein-associated phospholipase A 2 , an in ammatory marker, is an independent predictor of ischemic stroke and coronary heart disease. 8, 9 More importantly, Lp-PLA 2 is intimately associated with Lp(a) in atherosclerosis and cardiovascular disease. 10 Although previous studies examined the association of Lp(a) with risk of poor functional outcome in patients with ischemic stroke, 11,12,13 rare studies have conducted the relationship of Lp(a) and Lp-PLA 2 levels to stroke functional outcomes.
In this study, we aimed to evaluate the hypothesis that a high level of Lp(a) is associated with poor functional outcome and Lp(a) high/ Lp-PLA 2 high have a synergistic effect on functional outcomes in patients with ischemic stroke from the third China National Stroke Registry (CNSR-III) database.

Data availability
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Study population
We used the CNSR-III, a nationwide, prospective, multicenter, observational registration study based on etiology, imaging, and biology markers from patients with ischemic stroke and transient ischemic attack (TIA) between August 2015 and March 2018 in China. 14 Speci c information about the database has been described in detail in our previous studies. 14 In brief, a total of 15,166 consecutive patients from 171 hospitals were recruited, among which 93.3% with ischemic stroke (n = 14,146) and 6.7% with TIA (n = 1020). All patients were enrolled within 7 days after symptom onset. We included 10,422 individuals with complete information on plasma Lp(a) measurements. According to the principles mentioned in the Declaration of Helsinki, the ethics committees of Beijing Tiantan Hospital and all other recruited participating centers approved the study protocol. 15 Written informed consent was obtained from all participants (or guardians of participants) in this study.

Baseline data collection
An electronic data capture system by face-to-face interviews was used to collect CNSR-III clinical baseline data. The subsequent data were gathered from the registry database, including age, sex, body mass index (BMI), and smoking status; medical history of hypertension, diabetes mellitus, and lipid metabolism disorders; systolic blood pressure (SBP), fasting plasma glucose (FPG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), high-sensitivity Creactive protein (hsCRP), Lp(a), and lipoprotein-associated phospholipase A 2 ; stroke subtypes, classi ed as large artery atherosclerosis (LAA), and non-LAA according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria, 16 discharge medication rate of anticoagulant and antiplatelet drugs, and the National Institutes of Health Stroke Scale (NIHSS) score at admission.

Functional outcomes of stroke evaluation
The severity of ischemic stroke was evaluated using the NIHSS score at admission. Functional outcome was assessed with the modi ed Rankin scale (mRS) at 3 months and 1 year after stroke separately. The mRS scale ranges from 0 to 6, and the score parallels the severity of ischemic stroke. An mRS score of 0 was de ned as no residual stroke symptoms; 5, severe disability; and 6, death.

Laboratory analyses
Fasting blood specimens from 11,261 patients were collected using EDTA anticoagulation tubes within 1 day after admission and were centrifuged on-site within 2 h of collection to separate plasma for subsequent testing. Standard hospital assays were used on fresh plasma samples to measure plasma FPG, LDL-C, HDL-C, TG, hsCRP, and Lp-PLA 2 .

Lp(a) measurement
Lp(a) ELISA (Mercodia, Uppsala, Sweden) detects human Lp(a) and in terms of isoforms, is sizeindependent in terms of the kringle IV type 2 domain. The Mercodia ELISA is a solid phase two-site enzyme immunoassay and include a 5-point calibrator. The coefcient of variation (CV) was 7%.

Statistical analyses
Baseline characteristics categorized by Lp(a) levels were compared. Data on basic characteristics are presented as medians (interquartile ranges) for continuous variables. Categorical variables are presented as percentages. Nonparametric Wilcoxon test and chi-square test were used for comparisons of continuous and categorical variables between groups separately. The associations of Lp(a) with mRS at 3 months and 1 year were examined using a logistic regression model. We adjusted the potential confounders measured at baseline in the analysis. The model was adjusted for age, sex, BMI, FPG, LDL-C, HDL-C, TG, hsCRP, Lp-PLA 2 , TOAST subtype, and NIHSS score at admission. The strength of the associations was demonstrated using hazard ratios (HRs) with 95% con dence intervals (CIs). The sensitivity analysis was used to rule out the effects of recurrence and TIA on the association between the levels of Lp(a) and outcomes at 3 months. Two-sided p < 0.05 was considered to be statistically signi cant. The above statistical analyses were conducted using SAS version 9.4 (SAS Institute Inc., Cary, North Carolina).

Results
In the CNSR-III cohort of 15,166 patients with ischemic stroke or TIA, 12,603 patients from 171 sites were entered into a subgroup analysis of Lp(a) levels and functional outcomes. Blood samples from 11,261 of the 12,603 patients were collected and examined at the laboratory. According to the inclusion criteria, a total of 10,422 patients were included in the study, and the remaining 839 patients were excluded ( Figure  S1). In Table S1, the comparison of the included and excluded patients is shown. Compared with the excluded patients, the included patients showed signi cantly higher levels of LDL-C, Lp-PLA 2 , lower rate of current smoker, and higher discharge medication rates of anticoagulant and antiplatelet drugs.
Baseline characteristics Table 1 shows the baseline characteristics of the 10,422 individuals strati ed by Quartiles of baseline plasma Lp(a) levels. With the increment in plasma Lp(a) levels, patients tended to be older and had lower levels of BMI and TG, a higher proportion of LAA, and higher LDL-C, HDL-C, hsCRP, and Lp-PLA 2 .
Association between the levels of Lp(a) and stroke outcomes at 3 months and 1 year  (Table 3). We then used Lp-PLA 2 for subgroup analysis to further evaluate the relationship between Lp(a) levels and functional outcomes of stroke assessed using mRS ≥3 at 3 months. Table 4 shows concurrent analysis stratifying Lp(a) and Lp-PLA 2 into low vs. high levels (<median value vs. ≥median value). Good outcomes were observed among patients with Lp(a) low/ Lp-PLA 2 low group, whereas poor outcomes were observed among patients with Lp(a) high/ Lp-PLA 2 high group.

Sensitivity analysis
Association between the levels of Lp(a) and outcomes at 3 months after removing patients with recurrent stroke and TIA To rule out the effect of recurrence and TIA on the association between the levels of Lp(a) and outcomes at 3 months, we excluded the 3 months recurrent stroke and TIA population for further analysis. As shown in Table S2, in the unadjusted model, elevated levels of Lp(a) were positively correlated with the poor clinical outcomes of stroke as evaluated by mRS score ≥ 3 at 3 months. After adjustment for age, sex, BMI, FPG, LDL-C, HDL-C, TG, hsCRP, Lp-PLA 2 , TOAST subtype, and NIHSS score at admission, similar results were observed. Elevated levels of Lp(a) were positively correlated with the poor clinical outcomes of stroke as evaluated by mRS score ≥ 3 at 3 months.

Discussion
In the CNSR-III cohort study, we investigated the association between plasma Lp  11 Similarly, in a study conducted by Wang et al., who investigated 153 patients with acute ischemic stroke and 120 controls, an increased risk of poor functional outcomes was associated with Lp(a) levels. 12 In another study that recruited 100 consecutive patients with acute ischemic stroke and 120 controls, a positive relationship is suggested between Lp(a) levels and poorer long-term prognosis of stroke. 17 By contrast, Kooten et al. failed to nd any association of stroke prognosis with Lp(a) levels. 13 The association of stroke prognosis with Lp(a) level remains unclear. Therefore, a large sample cohort study is warranted to further clarify the relationship of ischemic stroke prognosis with Lp(a) levels. The current results demonstrated a positive correlation between the levels of Lp(a) and functional outcomes evaluated by mRS at 3 months and 1 year after stroke. To remove the effect of stroke recurrence and TIA on the conclusion, we further analyzed the relationship between Lp(a) levels and functional outcomes after excluding patients with stroke recurrence and TIA at 3 months, the relationship still existed. Our study con rmed a positive association between plasma Lp(a) levels and functional outcomes at 3 months and 1 year after ischemic stroke.
The mechanism through which Lp(a) levels are associated with functional outcomes of ischemic stroke remains unclear until now. In ammation runs through the onset, process, and progression of acute ischemic stroke. In acute ischemic stroke, microglial activation and cell death products trigger an in ammatory cascade, which damages the brain and affects functional outcomes. 7 The pathogenic effect of Lp(a) is partly due to its pro-in ammatory effect, which is harmful to the progression of ischemic stroke. The pro-in ammatory effect is mediated partially by its oxidized phospholipid content. 18 In addition, other molecules are related to the effect of Lp(a) pro-in ammatory. Our study revealed that Lp(a) levels and Lp-PLA 2 have a synergistic effect on functional outcomes. As a pro-in ammatory indicator, Lp-PLA 2 is mainly expressed on the surface of in ammatory cells enriched in plaques, and it promotes the secretion of in ammatory mediators by degrading oxidized phospholipids to cause endothelial dysfunction. 19 Endothelial dysfunction aggravates functional outcomes of ischemic stroke. 20,21 In a recent study, Schnitzler et al. demonstrated that oxidized phospholipids carried by Lp(a) drive endothelial in ammation. 18 Lp-PLA 2 may be considered as the synergistic molecule mediator for proin ammatory properties of Lp(a). We also found that with the increase in Lp(a) levels, hsCRP levels also increased. Combining the results of the present study with the conclusions of previous studies further indicates that elevated Lp(a) levels disrupt functional outcomes after ischemic stroke mainly through in ammatory pathways.
The present study is the largest by far to evaluate the clear association between plasma Lp(a) levels and functional outcomes after ischemic stroke. However, it has several limitations. First, we only measured Lp(a) at admission, and no serial measurement of Lp(a) levels was performed. For this reason, we could not conclude any causal relationship of functional outcomes with high Lp(a) levels. We only demonstrated a positive association of poor functional outcomes after ischemic stroke with high Lp(a) levels. Second, the study samples comprised Chinese individuals, limiting the application of its conclusions to other races and populations. Third, genetic data were lacking because of the observational nature of the study. Future studies are warranted to further investigate the effect of these factors on Lp(a) and functional outcomes in patients with ischemic stroke. Conclusions Elevated Lp(a) level is associated with poor functional outcomes evaluated by mRS at 3 months and 1 year after ischemic stroke. Lp(a) has a synergetic effect with Lp-PLA 2 on functional outcomes after ischemic stroke. Further studies should be carried out regarding the in ammation mechanism between increased Lp(a) levels and poor functional outcomes in patients with ischemic stroke. Declarations Ethics approval and consent to participate According to the principles mentioned in the Declaration of Helsinki, the ethics committees of Beijing Tiantan Hospital and all other recruited participating centers approved the study protocol. Written informed consent was obtained from all participants (or guardians of participants) in this study.

Consent for publication
All authors have approved this manuscript.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.