Biological Properties and Clinical Significance of Lipoprotein-Associated Phospholipase A2 in Ischemic Stroke

Ischemic stroke, which occurs following blockage of the blood supply to the brain, is a leading cause of death worldwide. Its main cause is atherosclerosis, a disease of the arteries characterized by the deposition of plaques of fatty material on the inner artery walls. Multiple proteins involved in the inflammation response have been identified as diagnosing biomarkers of ischemic stroke. One of these is lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme that can hydrolyze circulating oxidized phospholipids, generating proinflammatory lysophosphatidylcholine and promoting the development of atherosclerosis. In the last two decades, a number of studies have revealed that both the concentration and the activity of Lp-PLA2 are independent biomarkers of ischemic stroke. The US Food and Drug Administration (FDA) has approved two tests to determine Lp-PLA2 mass and activity for predicting stroke. In this review, we summarize the biological properties of Lp-PLA2, the detection sensitivity and limitations of Lp-PLA2 measurement, the clinical significance and association of Lp-PLA2 in ischemic stroke, and the prospects of therapeutic inhibition of Lp-PLA2 as an intervention and treatment.


Epidemiology of Stroke
Stroke is a disease caused by brain damage due to the interruption of the blood supply to the brain [1,2]. Based on data from the Global Burden of Disease Study 2019 (https://www .healthdata.org), stroke is the second leading cause of disability and mortality worldwide ( Figure 1). Stroke is normally classified into two subtypes: hemorrhagic stroke and ischemic stroke [3,4]. A hemorrhagic stroke occurs when a blood vessel ruptures and bleeds into the brain [3,4]. An ischemic stroke, which accounts for most cases (nearly 87%) [5,6], is characterized by a sudden loss of blood circulation due to blockage of an artery [5,6]. Current estimates indicate that stroke affects 13.7 million people and causes more than 5.5 million deaths globally each year [7,8]. Globally, about a quarter of the population experience an attack of stroke during their lifetimes [7,8]. In China, the stroke burden has increased over the past 40 years and the prevalence of stroke in Chinese adults aged ≥40 years is 2.58% according to the data from a 7-year (2013-2019) study [9]. Data from another study show that there were more than 3.94 million new stroke cases and 2 million death caused by stoke in China in 2019 [10].

Pathology and Risk Factors of Ischemic Stroke
In most cases, ischemic stroke is thromboembolic, with a common symptom of artery atherosclerosis that leads to fatty deposits and the accumulation of cholesterol plaque in the blood vessels (Figure 2(a)) [11,12]. Blood clots form in the arteries after the buildup of plaque, and they can block the blood flow to organs, especially the brain, causing ischemic stroke [11,12]. Small vessel disease is also an important cause of ischemic stroke, especially in Asian populations (Figure 2(a)) [13,14]. Small vessel disease damages the tiny blood vessels deep inside the brain, reducing blood flow and preventing brain cells from obtaining oxygen and nutrients [13,14]. Other conditions associated with the pathogenesis of ischemic stroke include atrial fibrillation, patent foramen ovale (PFO), and arterial dissection (Figure 2(a)) [15][16][17]. Atrial fibrillation is a type of irregular heart rhythm that contributes to thromboembolic stroke through stasis in the left atrium, causing embolization in the brain [15]. PFO is a hole between the two upper chambers of the heart and is present in >25% of the adult population [16]. Blood clots may form in PFO-containing patients when blood flows from one side of the heart to the other and up to the brain [16]. Cervical artery dissection is the most common cause of ischemic stroke in young adults [17]. Arterial dissection occurs after a tear, as blood enters the vessel wall, where it forms a clot and restricts the flow of blood to the brain, causing stroke [17].

Lp-PLA 2 in the Pathogenesis of Ischemic Stroke
Elevated levels of Lp-PLA 2 may lead to an increased risk for many diseases, such as coronary heart disease (CHD), myocardial infarction, and ischemic stroke [34,35]. In the clinical setting, elevated Lp-PLA2 levels have been used as a biomarker to predict the development of CHD in apparently healthy individuals and to predict ischemic stroke [34,35].
Other studies have also revealed that low levels of Lp-PLA 2 are associated with lipoprotein(a) and very lowdensity lipoproteins. The structure of human Lp-PLA 2 indicates that residues 114-120 and 360-368 are required for the association with membranes, while residues 192-204 are required for the association with HDL [29,31].
Unlike its binding in human serum, Lp-PLA 2 is mostly associated with HDL in the serum of other species, such as the rat, mouse, and pig, where the serum concentrations of LDL are lower than in humans [29]. A structure-based analysis demonstrated that the absence of Trp 115 and Leu 116 in the mouse Lp-PLA 2 may be the cause of varying binding efficiency [37]. The basal levels of Lp-PLA 2 also differ significantly among species. For example, the basal circulating concentration of Lp-PLA 2 is nearly 10-fold higher in mouse serum than in human serum [29,37]. This finding suggests that mimicking human diseases associated with increased Lp-PLA 2 may be difficult using animal models.

Determination of Lp-PLA 2 Concentration and Activity.
Lp-PLA 2 has been proposed as a biomarker for the evaluation of the risks of cardiovascular disease (CVD) and ischemic stroke [34,35]. Multiple methods have been developed to detect both Lp-PLA 2 concentration (mass) and Lp-PLA 2 activity in human serum. In 2003, the US Food and Drug Administration (FDA) approved the PLAC test to measure Lp-PLA 2 concentration using enzyme-linked immunosorbent assays (ELISAs) [38]. The normal range for the Lp-PLA 2 concentration in serum is less than 200 ng/mL, and people with Lp-PLA 2 levels of more than 200 ng/mL may be at greater risk of having a stroke [39]. The clinical application of these kits provides doctors with important evidence to diagnose the risks of CVD and ischemic stroke. In December 2014, the FDA approved another PLAC test that could measure Lp-PLA 2 activity [40]. Comparisons of the association of Lp-PLA 2 mass and activity in the same materials revealed correlation coefficients ranging from 0.36 to 0.86 and that the accuracy in predicting the risks of 3 Cardiovascular Therapeutics CVD and ischemic stroke was greater for Lp-PLA 2 activity than for Lp-PLA 2 concentration [41][42][43][44]. The risk of CHD and ischemic stroke is approximately 2.1-fold higher in people with PLAC activity over 225 nmol/min/mL than with PLAC activity less than 225 nmol/min/mL [45].
In 2018, Topbas et al. developed a liquid chromatography-tandem mass spectrometry-(LC-MS/MS-) based assay called "stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity" to quantify serum Lp-PLA 2 concentration [47]. The concentrations of serum Lp-PLA 2 at the peptide level were much higher (up to 8-   Cardiovascular Therapeutics fold) when determined by the SISCAPA-LC-MS/MS method than by the PLAC test [47]. Importantly, serum Lp-PLA 2 concentrations measured by SISCAPA-LC-MS/ MS show a good correlation with Lp-PLA 2 activity [47]. The reason for the discordance in serum concentrations of Lp-PLA2 determined by the standard ELISA method versus the SISCAPA-LC-MS/MS method may be due to a blockage of the sensitivity of the ELISA assay due to the interaction between Lp-PLA2 and lipoprotein [47]. The disassociation of Lp-PLA2 from lipoprotein by detergent can significantly increase the Lp-PLA 2 concentration determined using the PLAC test, while also improving the correlation between Lp-PLA 2 concentration and activity. Thus, improvement in the ELISA assay by supplementation with detergent may be beneficial for determining Lp-PLA 2 concentration in the clinic.
Recently, Chen et al. developed a highly sensitive timeresolved fluorescence immunoassay (TRFIA) to detect serum Lp-PLA 2 concentration in breast cancer patients [48]. TRFIA assay shows better characteristics than ELISA in terms of detection time and measurement range [48]. TRFIA only uses one-step assay and it can be finished within one hour. Moreover, TRFIA shows a 20% higher measurement range than that of ELISA [48].

Clinical Significance of Lp-PLA 2 in Ischemic Stroke.
The results of numerous large-scale observational studies have revealed that Lp-PLA 2 concentration and activity are both independent biomarkers of ischemic stroke. Most, but not all, observational studies have reported a positive association between Lp-PLA 2 concentration or activity and ischemic stroke (Table 1).
Persson et al. performed an urban population-based assay in 347 populations (195 patients with CHD and 152 with ischemic stroke) to characterize the association between Lp-PLA 2 mass or activity and the incidence of CHD and ischemic stroke [49]. After adjusting for age, sex, and cardiovascular factors, the researchers concluded that the elevated Lp-PLA 2 activity and mass were associated with an increased incidence of ischemic stroke but were not significantly related to CHD occurrence [49]. In the Northern Manhattan Study, Katan and colleagues investigated the association of   Lp-PLA 2 activity and mass IL-6 and YLK-40 were more apparent than Lp-PLA 2 mass and activity to predict recurrent stroke [58] 6

Cardiovascular Therapeutics
Cardiovascular Therapeutics Lp-PLA 2 mass and activity with stroke subtypes in a 1,946 stroke-free population [50]. After follow-up for 11 years, 151 participants (7.8%) experienced ischemic stroke at a mean age of 69 ± 10 years [50]. Statistical data revealed no association between either Lp-PLA 2 mass or activity levels and overall ischemic stroke risk [50]. However, Lp-PLA 2 mass, but not activity levels, was associated with strokes due to large artery atherosclerosis (LAA), but only among non-Hispanic Whites and not in other racial/ethnic groups [51]. The findings reported by Elkind et al. on the 467 ischemic stroke patients included in the population-based Northern Manhattan Stroke Study indicated that stroke patients with higher Lp-PLA 2 activity had an increased risk of recurrence after the first ischemic stroke [51]. However, Lp-PLA2 activity was not significantly affected by stroke severity [51]. Cao et al. conducted a study on 200 patients with acute ischemic stroke and 90 healthy controls to investigate the clinical utility of serum Lp-PLA2 activity [34]. They found that the Lp-PLA 2 concentration was much higher in acute ischemic stroke patients than in the control group and was positively correlated with the National Institutes of Health Stroke Scale score [34]. Lin and colleagues investigated the association between Lp-PLA 2 level and the early recurrence of vascular events in populations with a transient ischemic attack (TIA) and minor stroke [52]. After adjusting for age and gender, they concluded that Lp-PLA 2 levels were associated with the incidence of ischemic stroke, myocardial infarction, and death [52]. In a REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) study, Ridker et al. found that treatment with mineral oil caused a 25% higher risk than Icosapent Ethyl because mineral oil can increase the levels of Lp-PLA 2 , IL-1β, IL-6, oxidized LDL, CRP, and lipoprotein(a) [53]. Using a small population (169 patients), Wang reported that plasma Lp-PLA 2 concentration was associated with acute ischemic stroke in patients with atrial fibrillation [54].
In a systematic review study, Li et al. evaluated the association between Lp-PLA 2 and CHD in a huge population containing 30,857 participants [55]. The authors found no association between Lp-PLA 2 activity (or mass) and CHD mortality, whereas they found both Lp-PLA 2 activity and mass to be independent factors associated with the incidence of cardiovascular events [55]. In another meta-analysis, the Emerging Risk Factors Collaboration found that the Lp-PLA 2 level was an independent risk factor for cardiovascular events, with a hazard ratio of 1.12, after analyzing the data from 32,075 participants [56]. In 2007, the American Association of Clinical Endocrinologists (AACE) and the American College of Endocrinology (ACE) published guidelines for the management of dyslipidemia and prevention of cardiovascular disease, with the recommendation that patients at risk of cardiovascular or stroke be evaluated for serum Lp-PLA 2 activity and mass [57]. In a substudy of the Third China National Stroke Registry that included 10,472 participants, Li et al. found that IL-6 and YLK-40 were more apparent than CRP and Lp-PLA 2 mass and activity to predict recurrent stroke after ischemic stroke [58].

Inhibition of Lp-PLA 2
The important roles of Lp-PLA 2 in the pathogenesis of cardiovascular diseases and ischemic stroke suggest that it may be a potent therapeutic target. In vitro biochemical studies and in vivo animal experiments show that several Lp-PLA 2 inhibitors can significantly inhibit Lp-PLA 2 activity and prevent the progression of coronary atherosclerosis.
6.1. Darapladib. Darapladib (Figure 7(a)), discovered by Human Genome Sciences (HGS) and GlaxoSmithKline (GSK), is a potent inhibitor of Lp-PLA 2 [59]. In the first two rounds of clinical trials, darapladib showed promising inhibitory effects against Lp-PLA 2 activity and the inflammatory response [60]. However, GSK announced that darapladib had failed to meet Phase III endpoints in a trial (Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy, STABILITY) with 15,828 CHD patients, because this chemical did not achieve a statistically significant improvement in reducing the occurrence of cardiovascular death, myocardial infarction, and stroke [60]. In another clinical trial (Stabilisation Of pLaques usIng Darapladib-Thrombolysis InMyocardial Infarction 52 (SOLID-TIMI 52)) with 13,026 participants, darapladib (160 mg daily) also failed to reduce the risk of myocardial infarction, urgent coronary revascularization, or death from CHD [61]. The failed clinical trials for darapladib raise concerns regarding the suitability of using Lp-PLA 2 as a drug target. Instead, it may only be useful as a biomarker for cardiovascular diseases and ischemic stroke.
6.2. Rilapladib. Rilapladib (Figure 7(b)) is a small-molecule drug identified from the collaboration between HGS and GSK [62]. A randomized and double-blind clinical trial under phase 2a provided preliminary evidence that the inhibition of Lp-PLA 2 by administration of 250 mg rilapladib may attenuate the progression of Alzheimer's disease [62]. However, this trial recruited only 124 participants, and the primary outcomes were evaluated at the 24 th week [62]. The preliminary findings require repetition in a larger population and for a longer duration. Although rilapladib is an inhibitor of Lp-PLA 2 , animal experiments and human clinical trials are still lacking to confirm the effectiveness of rilapladib in the prevention of ischemic stroke.
6.3. Other Compounds. Apart from darapladib and rilapladib, several other compounds also show strong abilities to inhibit Lp-PLA 2 activity. For example, compound 1 (Figure 7(c)) interacts with the oxyanion hole in Lp-PLA 2 through two bonds to the backbone amides of Phe 274 and Leu 153 [29]. Compound 2 (Figure 7(d)), a bioisostere of darapladib, shows a similar binding mode to that of darapladib, but it has improved physicochemical properties [63]. Huang et al. used a covalent fragment-based approach to identify a highly selective compound 3 (Figure 7(e)), which showed a much higher (130,000-fold) potency and 3,900-fold selectivity compared to a covalent fragment [32]. Three other compounds (4, 5, and 6) (Figures 5(f)-5(h)) also show promising potency for interaction with Lp-PLA 2 [64][65][66]. Although in vitro biochemical studies and crystal structure analyses reveal that   Cardiovascular Therapeutics these compounds significantly inhibit Lp-PLA 2 activity, these small molecules have not yet been utilized in any human clinical trials. Thus, whether these compounds have potent abilities to improve the outcome of ischemic stroke is unknown.

Conclusions and Prospects
The global prevalence of ischemic stroke causes mortality and disability in a quarter of the world's population, particularly for people in lower-income and lower-middleincome countries [6]. Stroke can be caused by many risk factors, such as aging, obesity, high blood pressure, alcohol consumption, and lacking physical activity. However, it is still lacking urgent implementation of effective primary prevention strategies at present, suggesting that the stroke burden will continue to increase in the future. To decrease the public health burden caused by stroke, two aspects of work need to be carried out simultaneously. One is to develop reliable stroke biomarkers and efficient detection methods and then screen the general population to predict stoke risk. The second aspect is to develop more effective strategies for stroke treatment in clinical. In response to this, a very important strategy is to conduct in-depth research on the mechanisms of stroke occurrence and development, identify some key therapeutic targets, and develop drugs against these targets. Evaluation of ischemic stroke risks and treatment are both important in decreasing the global burden [3,5,8]. Accumulated evidence has proven that both Lp-PLA 2 activity and mass are independent biomarkers of ischemic stroke and cardiovascular disease [29]. The US FDA has approved two PLAC assays to measure Lp-PLA 2 concentration and activity. Some clinical studies have revealed that ischemic stroke shows less association with Lp-PLA 2 concentration than with Lp-PLA 2 activity. Thus, the supplementation of detergent in standard PLAC mass assays to disassociate the interactions of Lp-PLA 2 and lipoprotein may increase the sensitivity and accuracy of ELISA assays. The development of other simple and accurate Lp-PLA 2 detection methods may also improve the possibility of predicting ischemic stroke risk.
Besides their association with ischemic stroke, elevated Lp-PLA 2 levels are also common in some other diseases, such as cardiovascular diseases and Alzheimer's disease [29]. Recently, Lp-PLA 2 is suggested to be a biomarker of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because it is correlated with most of the known comorbidities and complications of this disease [67]. Thus, the ischemic stroke risk cannot be judged solely on the basis of the elevated levels and activities of Lp-PLA 2 . Published studies have paid little attention to the correlations between Lp-PLA 2 activity (or mass) and other ischemic stroke biomarkers, including CRP, MMPs, IL-6, TNF-α, VCAM1, ICAM1, S100B, vWF, BNGF, and MCP1. Panels that combine Lp-PLA 2 activity (or mass) with these biomarkers may increase the diagnostic accuracy of ischemic stroke risk.
Although different research groups have resolved the crystal structures of Lp-PLA 2 , both alone and when bound with other inhibitors, medicines that can target Lp-PLA 2 are still lacking for the treatment of ischemic stroke and other diseases associated with the elevations of Lp-PLA 2 activity or mass. The failure of the clinical trial of darapladib reminds us to reconsider whether Lp-PLA 2 is truly a suitable target. The observation that PLA2G7, the encoding gene for Lp-PLA 2 , is also overexpressed in the pathogenesis of cardiovascular diseases and ischemic stroke suggests that dissection of the upstream regulatory mechanism of PLA2G7 may benefit the development of therapeutic strategies to decrease Lp-PLA 2 activity and mass.

Data Availability
The data underlying the present study are available on request (corresponding author).

Conflicts of Interest
The authors declare no conflict of interest, financial or otherwise.   9 Cardiovascular Therapeutics Shuang Zhang and Shuchun Huang contributed equally to this paper.