- Email: [email protected]
Lipoprotein-associated Phospholipase A2 during the Hyperacute Stage of Ischemic and Hemorrhagic Strokes
Lipoprotein-associated Phospholipase A2 during the Hyperacute Stage of Ischemic and Hemorrhagic Strokes Charlotte Rosso, MD, PhD,*†‡ David Rosenbaum, MD,xk Christine Pires, MSc,‡ Corinne Cherfils, PharmD,{ Nabil Koujah, PharmD,{ Fouzi Mestari, PharmD,{ Emeline Gillet, MD,{ Sophie Crozier, MD,‡ Melika Sahli-Amor, MD,# Yves Samson, MD,*†‡ Dominique Bonnefont-Rousselot, PharmD,{**1 and Randa Khani-Bittar, PharmD{††1
Background: The objectives of the study were to compare lipoprotein-associated phospholipase A2 (Lp-PLA2) levels in a prospective cohort including both ischemic and hemorrhagic strokes at the hyperacute phase, and to investigate if these levels were associated with stroke severity. Materials and methods: Lp-PLA2 mass and activity were measured during the first 6 hours of symptom onset before any therapeutic intervention. The Lp-PLA2 level was analyzed by comparing the mass and activities in ischemic strokes and spontaneous intracerebral hemorrhages (ICH). Correlations between Lp-PLA2 levels and clinical scores as well as stroke volumes were made. The temporal evolution of Lp-PLA2 during the first week was analyzed in ischemic stroke patients. Results: Lp-PLA2 mass was higher in ICH than in ischemic stroke (P 5 .001). Lp-PLA2 activity at admission correlated with initial and follow-up stroke volume in ICH (P 5 .003 and P 5 .004, respectively) but not in ischemic stroke. None of the measurements correlated with clinical severity for either diagnosis. Lp-PLA2 mass decreased during the first week after the use of statins in ischemic stroke, whereas the activity remained stable. Conclusions: Lp-PLA2 mass is higher in ICH compared with ischemic stroke during the hyperacute stage. Lp-PLA2 activity is associated with stroke volume in ICH but not in ischemic stroke. This suggests that Lp-PLA2 mass and activity could provide different information in the hyperacute stage of stroke. Key Words: Lipids and lipoprotein—cerebral infarction—cerebral hemorrhage—stroke—statins. Ó 2014 by National Stroke Association
Introduction Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory biomarker that has been described as an independent risk marker for recurrent events in ischemic
From the *CRICM—Centre de Recherche de l’Institut du Cerveau et de la Moelle epiniere, UPMC Paris 6, Inserm, U975, CNRS, UMR; †COGIMAGE, UPMC Paris 6; ‡APHP, Urgences Cerebro-Vasculaires, H^ opital Pitie-Salp^etriere; xUnite de prevention des maladies cardiovasculaires, H^ opital Pitie-Salp^etriere; kLaboratoire d’imagerie fonctionnelle, INSERM U678, UPMC Paris 6; {APHP, Service de Biochimie Metabolique, H^ opitaux Universitaires Pitie-Salp^etriereCharles Foix; #APHP, Service de Neuroradiologie, H^ opital PitieSalp^etriere; **EA 4466, Departement de Biologie Experimentale, Metabolique et Clinique, Faculte de Pharmacie, Universite Paris
stroke and myocardial infarction.1 Lp-PLA2 plays a role in the pathophysiology of cerebrovascular disease, particularly in strokes of atherosclerotic ischemic etiology,2 because Lp-PLA2 is linked to oxidized low-density
Descartes, Sorbonne Paris Cite; and ††UPMC Universite Paris 06, Paris, France. Received September 12, 2013; revision received November 24, 2013; accepted November 27, 2013. Address correspondence to Charlotte Rosso, MD, PhD, APHP, Urgences Cerebro-Vasculaires, H^ opital Pitie-Salp^etriere, 47-83 Bd de l’H^ opital, 75013 Paris, France. E-mail: [email protected]. 1 These authors contributed equally to this work. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.11.024
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lipoprotein (LDL)-cholesterol and accumulates in the atherosclerotic plaque. Recently, beyond this role in the prevention of cardiac or cerebral events, Lp-PLA2 mass and activity have been measured during the hyperacute phase of ischemic stroke in a population treated by recombinant tissue plasminogen activator (rtPA).3 In that study, the mass of Lp-PLA2 predicted the risk of persistent intracranial occlusion; thus, it could be a surrogate of stroke severity. Although increasing evidence suggests that LpPLA2 could be a biomarker of events in ischemic strokes, Lp-PLA2 mass and activity have not thus far been tested in patients with hemorrhagic strokes and compared with the ischemic strokes during the hyperacute stage. Therefore, in this study, the primary objective was to compare the Lp-PLA2 mass and activity during the hyperacute phase (within the first 6 hours) after focal symptom onset in patients with ischemic stroke and spontaneous intracerebral hemorrhages (ICH). The second objective was to determine whether the Lp-PLA2 mass and activity during this period was a biomarker of the severity and outcome in these 2 populations. The hypotheses were that Lp-PLA2 levels will be higher in ischemic strokes than in hemorrhagic strokes and Lp-PLA2 levels will be positively correlated with stroke volumes.
scanner (General Electric Medical System, Milwaukee). The MRI protocol included 3 sequences: diffusionweighted imaging (DWI), Fluid Attenuated Inversion Recovery (FLAIR) and intracranial time-of-flight magnetic resonance angiography. MRI parameters are provided in the supplementary online material. For ischemic stroke patients, MRI was repeated at day 1. The initial and final infarct volumes were computed on the initial and follow-up DWI.4 The initial infarct volume was defined as the abnormal bright area on the initial DWI image set (bvalue 5 1000 s/mm2) and was measured using interactive manual outlining. The final infarct volume was measured using the same method from the follow-up DWI. For hemorrhagic stroke, patients were imaged using computed tomography just after MRI to confirm the diagnosis and enable size measurements. The volumes were assessed on the initial and follow-up computed tomography scans.5 The volume was approximated using the following formula: A 3 B 3 C/2, where A is the largest diameter of the hyperdensity, B is its perpendicular diameter, and C is the number of slices multiplied by the slice thickness.
Blood Sample Collection and Analysis
Methods Patients We recruited patients prospectively who were referred to the stroke unit between February 2011 and June 2012 based on the following criteria1: a focal deficit compatible with the diagnosis of stroke,2 an initial magnetic resonance imaging (MRI) which was performed within the first 6 hours of symptom onset and confirmed the diagnosis of ischemic or hemorrhagic stroke,3 and a blood sample with Lp-PLA2 measurements taken before the MRI. The National Institutes of Health Stroke Scale (NIHSS) was rated on admission and at days 1 and 7, and the modified Rankin scale (mRS) was scored at 3 months. The final diagnosis for each patient was made by stroke neurologists and confirmed by brain imaging. All patients had an ultrasound echography of the cervical arteries. Stroke etiology was determined at follow-up according to the following classification: atherosclerotic (significant atheroma, ie, stenosis on the cervical arteries or plaques .4 mm in the aortic arch), cardiogenic (such as atrial fibrillation), lacunar infarction, cryptogenic stroke, and others (such as cervical artery dissection). All imaging and clinical data were generated during the routine clinical workup of the patients in our stroke center. The local ethics committee approved the study.
Brain Imaging Methods MRI was performed during the first 6 hours of symptom onset using a 3-T whole-body MR General Electric
Peripheral venous blood samples were obtained from all patients before the MRI and any treatment. When an ischemic stroke was diagnosed, the samples on days 1 and 7 were repeated. Fresh venous blood was collected in gel-containing Vacutainer tubes (Becton-Dickinson, Plymouth, UK) and then centrifuged at 4500 rpm at 4 C for 10 minutes within 2 hours after withdrawal. Serum was aliquoted into many aliquots and stored at 280 C until analyses.6,7 Lipid Parameters Fresh serum lipids were analyzed using routine methods on a Konelab 30i analyzer (Thermo Electron Corporation, Waltham, Massachusetts). Triglycerides were measured using an automated enzymatic method (Diasys Diagnostic Systems GMBH, Condom, France).8 Total cholesterol and direct high-density lipoprotein (HDL)cholesterol were determined based on recommendations of ARCOL (Comite Franc¸ais de Coordination des Recherches sur l’Atherosclerose et le Cholesterol) using automated enzymatic methods9-11 (Konelab Thermo Fisher Scientifics Cergy-Pontoise, France). LDL-cholesterol was calculated using the Friedewald formula.12 Measurement of Lp-PLA2 Mass and Activity Frozen, collected serum aliquots were thawed at 4 C and assayed for Lp-PLA2 mass and activity. Lp-LA2 mass was assessed using the Plac test enzyme-linked immunosorbent assay kit (Diadexus Inc, South San Francisco, CA) that uses 2 monoclonal antibodies that are
PROGNOSTIC VALUE OF Lp-PLA2 IN ACUTE STROKE 13
specific for Lp-PLA2. Lp-PLA2 activity was performed using an automated colorimetric method on a Konelab 30i analyzer using a rate reaction assay with 1myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine as a substrate (Diadexus Inc; Eurobio, France). The longterm stability of Lp-PLA2 has been demonstrated in frozen serum samples. Activity was expressed as nanomoles of platelet-activating factor hydrolyzed per minute per milliliter of serum (nmoL/min/mL). All assays were performed in the Department of Metabolic Biochemistry of Pitie-Salp^etriere Charles Foix Hospital.
Data Analysis and Statistical Methods Descriptive data are presented as the mean 6 standard deviation or median and interquartile range (IQR). The distributions of Lp-PLA2 mass and activity were normal. Proportions were compared using the chi-squared test. One-way analysis of variance (ANOVA) with the diagnosis (ischemic strokes versus ICH) as a between-subject factor and the LDL-level and the level of atheroma as covariates was used to compare the Lp-PLA2 mass and activities. Atheroma severity was scored by a trained cardiologist as follows: 0 for no atheroma at all, 1 for plaques, 2 for stenosis under 70% ECST (European Carotid Surgery Trial) or 60% NASCET (North American Symptomatic Carotid Endarterectomy Trial), and 3 for more severe stenosis. The temporal evolution of Lp-PLA2 levels in ischemic strokes was investigated using repeated-measures ANOVA with the Lp-PLA2 mass or activity as the dependent variable and the time (days 0, 1, and 7) as the within-subject factor. Post hoc t tests were then performed. Correlations were computed using the bilateral Pearson correlation coefficients (MedCalc Package, version 9.3.2.0, Belgium).
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score at day 1 or 7. There was no significant correlation with the 3-month outcome assessed using the modified Rankin scale score. The initial infarct volume or final infarct volume did not correlate with either the mass or activity. However, Lp-PLA2 mass correlated with activity (r 5 .380, 95% CI: .344-.655, P 5 .005). The temporal evolution analysis (Fig 2) indicated that Lp-PLA2 activity remained stable over time (P 5 .60) whereas Lp-PLA2 mass decreased (F(1,60): 4.06, P 5 .021). Post hoc t tests revealed that Lp-PLA2 mass was lower at day 7 compared with day 1 (median at day 1: 285 mg/L, IQR: 232-323 versus median at day 7: 257, IQR: 209-320; P 5 .007). LDL-cholesterol also decreased between days 1 and 7 (median at day 1: 1.14 g/L, IQR: .78-1.47 versus median at day 7: .90 g/L, IQR: .70-1.20; P 5 .0002). Statins were provided to 30% of the included population before admission, 37% (20 of 54) at day 1 and 61% (33 of 54) at day 7. We finally divided our population according to the etiology of stroke (atherosclerotic n 5 15, cardiogenic n 5 18, lacunar n 5 2, cryptogenic n 5 11, and others n 5 8), and we have examined the differences in both Lp-PLA2 mass and activity between atherosclerotic infarction and other etiologies. No differences between the subgroups were found (Supplementary figure 1). For ICH, Lp-PLA2 activity measured during the first 6 hours of stroke onset correlated with hematoma volume at admission (r 5 .740, 95% CI: .320-.917, P 5 .003) and at follow-up (r 5 .731, 95% CI: .303-.914, P 5.004; Fig 3). LpPLA2 mass did not correlate with the volume at admission and follow-up. No significant correlation was found for either Lp-PLA2 mass and activity with clinical score during the length of stay or for the 3-month outcome.
Discussion Results Sixty-eight patients were recruited prospectively after sudden-onset focal deficits. The diagnoses were ischemic stroke (79%, n 5 54) and ICH (21%, n 5 14). The median time delay from stroke onset to brain imaging was 3.3 hours (IQR: 1.9-4.2 hours). Table 1 shows the clinical and biological characteristics of the entire population and subgroups.
Comparison of Lp-PLA2 Levels Between Ischemic versus ICH The ANOVA test revealed a global overall significant difference in Lp-PLA2 mass based on the diagnosis (F(1,64): 10.7, P 5 .002) but not for the Lp-PLA2 activity (P 5 .38). Post hoc analyses revealed that Lp-PLA2 mass was higher in hemorrhagic than in ischemic stroke (Fig 1).
Prognostic Value in Stroke Patients For ischemic stroke patients, Lp-PLA2 mass and activity at admission (,6 hours of stroke onset) did not correlate with the NIHSS score at admission or the NIHSS
In our study, we observed that Lp-PLA2 mass differed between stroke diagnoses (ICH versus ischemic strokes). Moreover, Lp-PLA2 activity (,6 hours) correlated with hematoma volumes at admission and follow-up. Lp-PLA2 levels were not associated with greater clinical severity or larger ischemic stroke volumes as judged using brain imaging. Finally, in ischemic stroke, the Lp-PLA2 mass decreased during the first week when patients were provided with statins,6 whereas its activity remained stable. Lower Lp-PLA2 mass in ischemic compared with hemorrhagic strokes was a surprising finding. The first reason is that previous studies reported a higher risk of spontaneous hemorrhagic stroke in persons with low cholesterol levels.14,15 Therefore, we theorize that lower Lp-PLA2 levels may also characterize this ICH subpopulation. Second, Lp-PLA2 has been implicated in atherosclerotic processing and should thus be more involved in the physiopathology of ischemic stroke. Indeed, the presence of oxidized phospholipids and increased Lp-PLA2 has been also demonstrated in experimental nonatherosclerotic cerebral ischemia.16,17 It should be also
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Table 1. Descriptive characteristics Clinical and biological characteristics Age (y) Gender % (n) Admission NIHSS 3-month mRS 0-1% (n) Lp-PLA2 mass (mg/L) Lp-PLA2 activity (nmol/min/ml) LDL level (g/L) HDL level (g/L) Total cholesterol level (g/L) Triglycerides (g/L) Known hypertension % (n) Known diabetes % (n) Statins at admission % (n) Tobacco use % (n)
All (n 5 68)
Ischemic stroke (n 5 54)
Hemorrhagic stroke (n 5 14)
P value
70 55-81 56% (38) 13 6-19 28% (19) 307 249-360 163 130-194 1.13 .76-1.40 .51 .41-.67 1.91 1.52-2.25 1.05 .86-1.36 66% (45) 18% (12) 28% (19) 18% (12)
70 55-83 46% (25) 10 3-17 31% (17) 291 248-342 160 124-186 1.15 .85-1.41 .51 .41-.68 1.9 1.50-2.30 1.10 .90-1.30 65% (35) 17% (9) 30% (16) 19% (10)
73 55-81 92% (13) 16 11-20 14% (2) 406 290-430 183 152-211 0.93 .71-1.28 .53 .42-.64 1.7 1.50-2.10 .90 .70-1.80 71% (10) 21% (3) 21% (3) 14% (2)
.98 .005 .054 .35 .001 .10 .20 .72 .29 .38 .91 .96 .73 .96
Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; Lp-PLA2, lipoprotein-associated phospholipase A2; mRS, modified Rankin scale; NIHSS, National Institutes of Health Stroke Scale. Data are shown as the median and interquartile range.
noticed that the initial blood sample analyzed in our cohort was taken in the first 6 hours of stroke onset. LpPLA2 is thought to have a half-life of about a few days, related to the LDL particles half-life. This rather long period is not compatible with an acute increase of LpPLA2. However, the exact half-life of Lp-PLA2 is not known yet. Because Lp-PLA2 is considered a specific marker of vascular inflammation, it may also be elevated in ICH, for example, as a marker of hypertensive vasculopathy. This finding is consistent with the study of Romero et al.18 on APOε2- or ε4-allele carrying patients. In that study, Lp-PLA2 levels were associated with the deep loca-
tion of microbleeds, which is typical of a pattern of hypertensive angiopathy. It is possible that Lp-PLA2 itself plays a role in the rupture of vessel-wall integrity via inflammation processes.19,20 Indeed, a reason that might explain the increase in Lp-PLA2 in ICH patients could be secondary changes of the brain tissue around the hematoma. Actually, as minutes after onset of ICH, the extravasated blood components and the associated molecular patterns released from necrotic and damaged tissue impose a strong proinflammatory state in the adjacent viable brain cells. Therefore, it may be possible that the increase in LpPLA2 in ICH may be because of the infiltration of white cells with a proinflammatory phenotype around the
Figure 1. Lp-PLA2 activity (A) and mass (B) measured at admission for intracranial cerebral hemorrhages (ICH) and ischemic strokes (IS). Lp-PLA2, lipoprotein-associated phospholipase A2.
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Figure 2. Temporal evolution of Lp-PLA2 mass, activity, and LDL-cholesterol level in the ischemic stroke patients from admission to day 7. LDL, low-density lipoprotein; LpPLA2, lipoprotein-associated phospholipase A2. *p ,.05
hematoma.21,22 The same hypothesis could also explain the correlation between Lp-PLA2 and the size of the hematoma. However, the mechanisms underlying this association remain unknown and must be specified. We did not find any correlation between clinical stroke severity and Lp-PLA2 levels (mass or activity) during the first week of stroke. These results are consistent with those of Delgado et al.,3 where no correlation between baseline clinical severity, as assessed using NIHSS score, and Lp-PLA2 was reported in a population of rt-Pa-treated patients. Moreover, in the PROVE-IT (Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis In Myocardial Infarction) trial,23 the baseline Lp-PLA2 measured immediately after the coronary event did not correlate with the risk of recurrence whereas the measurements after 1 month did. Finally, the main reason why Lp-PLA2 mass or activity did not differ between stroke etiology groups could be the relatively small size of our population. However, Lp-PLA2 mass and activity were high in our population, in comparison with controls. In healthy controls,6 mean Lp-PLA2 mass level was 163 6 43 mg/L (166 6 45 mg/L in men and 159 6 39 mg/L in women). For ICH, Lp-PLA2 was a biological marker of severity during the acute stage. In particular, Lp-PLA2 activity
Figure 3. Regression analysis. Plot of the initial (A) and follow-up ICH volume (B) as a function of the Lp-PLA2 activity at admission. A higher Lp-PLA2 activity at admission indicates a greater ICH volume. Lp-PLA2, lipoprotein-associated phospholipase A2.
correlated with hematoma volume at admission and at follow-up: higher Lp-PLA2 activity indicated a larger volume for the intracerebral hemorrhage. Because hematoma volume is a surrogate of the clinical outcome in ICH24 and correlates with Lp-PLA2, Lp-PLA2 may also be predictive of the outcome. Our small sample size and our rather short follow-up may underlie the fact that we did not observe this finding in our study. Moreover, the correlation was mainly driven by the patients with bigger volumes of ICH. However, when removing the patient with the biggest ICH volume, the correlation between Lp-PLA2 activity at admission and hematoma volume at day 1 was still significant (correlation coefficient: .614, P 5 .02). These preliminary findings must be confirmed in a larger cohort to determine whether they can be used in a clinical setting. This study suffers from limitations. Because our primary hypothesis was that Lp-PLA2 was significantly higher and meaningful in ischemic stroke, we did not plan assessments of temporal evolution in the ICH group. It would be interesting to study Lp-PLA2 levels in this latter group to determine whether the levels decrease with the resorption of the hematoma. Secondly, our sample size was too small to enable subgroup analyses, for example, between lobar and deep hemorrhages, or to
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compare different ICH etiologies. Moreover, whether increased Lp-PLA2 is causal or secondary to ICH needs to be determined by basic science experimentations and prospective clinical trials. However, such a thing has never been signaled in all the previous studies on LpPLA2 in primary or secondary prevention and needs further investigations.
Conclusions Our main result is that LpPLA2 mass is higher in ICH than in ischemic stroke. Moreover, we showed that LpPLA2 activity, as measured during the first 6 hours of stroke onset, correlated with hematoma volume at follow-up and could serve as prognostic markers. This suggests that LpPLA2 could provide different information at the hyperacute stage of stroke, especially in ICH. More studies on Lp-PLA2 mass and activity are required in brain hemorrhages to detail and confirm our results. Acknowledgments: The authors thank Franc¸oise Toublan for her valuable assistance with the organization of this study. The Lp-PLA2 kits were provided by DiaDexus. Conflicts of interest: Although Lp-PLA2 measurement kits were provided by Diadexus, at no cost, Diadexus played no role in the study design or statistical analyses. David Rosenbaum has received speaking fees from Diadexus.
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Supplementary Data
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Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013. 11.024.
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References 1. Gorelick PB. Lipoprotein-associated phospholipase A2 and risk of stroke. Am J Cardiol 2008;101:34F-40F. 2. Nambi V, Hoogeveen RC, Chambless L, et al. Lipoprotein-associated phospholipase A2 and high-sensitivity C-reactive protein improve the stratification of ischemic stroke risk in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2009;40:376-381. 3. Delgado P, Chac on P, Penalba A, et al. Temporal profile and prognostic value of Lp-PLA2 mass and activity in the acute stroke setting. Atherosclerosis 2012;220:532-536. 4. Rosso C, Hevia-Montiel N, Deltour S, et al. Prediction of infarct growth based on apparent diffusion coefficients: penumbral assessment without intravenous contrast material. Radiology 2008;250:184-192. 5. Hussein HM, Tariq NA, Palesch YY, et al, ATACH Investigators. Reliability of hematoma volume measurement at local sites in a multicenter acute intracerebral hemorrhage clinical trial. Stroke 2013;44:237-239. 6. Charniot JC, Khani-Bittar R, Albertini JP, et al. Interpretation of lipoprotein-associated phospholipase A2 levels is influenced by cardiac disease, comorbidities, extension of atherosclerosis and treatments. Int J Cardiol 2013; 168:132-138. 7. Charniot JC, Khani-Bittar R, Albertini JP, et al. Serial lipoprotein-associated phospholipase A2 assays: prog-
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Background: The objectives of the study were to compare lipoprotein-associated phospholipase A2 (Lp-PLA2) levels in a prospective cohort including both ischemic and hemorrhagic strokes at the hyperacute phase, and to investigate if these levels were associated with stroke severity. Materials and methods: Lp-PLA2 mass and activity were measured during the first 6 hours of symptom onset before any therapeutic intervention. The Lp-PLA2 level was analyzed by comparing the mass and activities in ischemic strokes and spontaneous intracerebral hemorrhages (ICH). Correlations between Lp-PLA2 levels and clinical scores as well as stroke volumes were made. The temporal evolution of Lp-PLA2 during the first week was analyzed in ischemic stroke patients. Results: Lp-PLA2 mass was higher in ICH than in ischemic stroke (P 5 .001). Lp-PLA2 activity at admission correlated with initial and follow-up stroke volume in ICH (P 5 .003 and P 5 .004, respectively) but not in ischemic stroke. None of the measurements correlated with clinical severity for either diagnosis. Lp-PLA2 mass decreased during the first week after the use of statins in ischemic stroke, whereas the activity remained stable. Conclusions: Lp-PLA2 mass is higher in ICH compared with ischemic stroke during the hyperacute stage. Lp-PLA2 activity is associated with stroke volume in ICH but not in ischemic stroke. This suggests that Lp-PLA2 mass and activity could provide different information in the hyperacute stage of stroke. Key Words: Lipids and lipoprotein—cerebral infarction—cerebral hemorrhage—stroke—statins. Ó 2014 by National Stroke Association
Introduction Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory biomarker that has been described as an independent risk marker for recurrent events in ischemic
From the *CRICM—Centre de Recherche de l’Institut du Cerveau et de la Moelle epiniere, UPMC Paris 6, Inserm, U975, CNRS, UMR; †COGIMAGE, UPMC Paris 6; ‡APHP, Urgences Cerebro-Vasculaires, H^ opital Pitie-Salp^etriere; xUnite de prevention des maladies cardiovasculaires, H^ opital Pitie-Salp^etriere; kLaboratoire d’imagerie fonctionnelle, INSERM U678, UPMC Paris 6; {APHP, Service de Biochimie Metabolique, H^ opitaux Universitaires Pitie-Salp^etriereCharles Foix; #APHP, Service de Neuroradiologie, H^ opital PitieSalp^etriere; **EA 4466, Departement de Biologie Experimentale, Metabolique et Clinique, Faculte de Pharmacie, Universite Paris
stroke and myocardial infarction.1 Lp-PLA2 plays a role in the pathophysiology of cerebrovascular disease, particularly in strokes of atherosclerotic ischemic etiology,2 because Lp-PLA2 is linked to oxidized low-density
Descartes, Sorbonne Paris Cite; and ††UPMC Universite Paris 06, Paris, France. Received September 12, 2013; revision received November 24, 2013; accepted November 27, 2013. Address correspondence to Charlotte Rosso, MD, PhD, APHP, Urgences Cerebro-Vasculaires, H^ opital Pitie-Salp^etriere, 47-83 Bd de l’H^ opital, 75013 Paris, France. E-mail: [email protected]. 1 These authors contributed equally to this work. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.11.024
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 4 (April), 2014: pp e277-e282
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lipoprotein (LDL)-cholesterol and accumulates in the atherosclerotic plaque. Recently, beyond this role in the prevention of cardiac or cerebral events, Lp-PLA2 mass and activity have been measured during the hyperacute phase of ischemic stroke in a population treated by recombinant tissue plasminogen activator (rtPA).3 In that study, the mass of Lp-PLA2 predicted the risk of persistent intracranial occlusion; thus, it could be a surrogate of stroke severity. Although increasing evidence suggests that LpPLA2 could be a biomarker of events in ischemic strokes, Lp-PLA2 mass and activity have not thus far been tested in patients with hemorrhagic strokes and compared with the ischemic strokes during the hyperacute stage. Therefore, in this study, the primary objective was to compare the Lp-PLA2 mass and activity during the hyperacute phase (within the first 6 hours) after focal symptom onset in patients with ischemic stroke and spontaneous intracerebral hemorrhages (ICH). The second objective was to determine whether the Lp-PLA2 mass and activity during this period was a biomarker of the severity and outcome in these 2 populations. The hypotheses were that Lp-PLA2 levels will be higher in ischemic strokes than in hemorrhagic strokes and Lp-PLA2 levels will be positively correlated with stroke volumes.
scanner (General Electric Medical System, Milwaukee). The MRI protocol included 3 sequences: diffusionweighted imaging (DWI), Fluid Attenuated Inversion Recovery (FLAIR) and intracranial time-of-flight magnetic resonance angiography. MRI parameters are provided in the supplementary online material. For ischemic stroke patients, MRI was repeated at day 1. The initial and final infarct volumes were computed on the initial and follow-up DWI.4 The initial infarct volume was defined as the abnormal bright area on the initial DWI image set (bvalue 5 1000 s/mm2) and was measured using interactive manual outlining. The final infarct volume was measured using the same method from the follow-up DWI. For hemorrhagic stroke, patients were imaged using computed tomography just after MRI to confirm the diagnosis and enable size measurements. The volumes were assessed on the initial and follow-up computed tomography scans.5 The volume was approximated using the following formula: A 3 B 3 C/2, where A is the largest diameter of the hyperdensity, B is its perpendicular diameter, and C is the number of slices multiplied by the slice thickness.
Blood Sample Collection and Analysis
Methods Patients We recruited patients prospectively who were referred to the stroke unit between February 2011 and June 2012 based on the following criteria1: a focal deficit compatible with the diagnosis of stroke,2 an initial magnetic resonance imaging (MRI) which was performed within the first 6 hours of symptom onset and confirmed the diagnosis of ischemic or hemorrhagic stroke,3 and a blood sample with Lp-PLA2 measurements taken before the MRI. The National Institutes of Health Stroke Scale (NIHSS) was rated on admission and at days 1 and 7, and the modified Rankin scale (mRS) was scored at 3 months. The final diagnosis for each patient was made by stroke neurologists and confirmed by brain imaging. All patients had an ultrasound echography of the cervical arteries. Stroke etiology was determined at follow-up according to the following classification: atherosclerotic (significant atheroma, ie, stenosis on the cervical arteries or plaques .4 mm in the aortic arch), cardiogenic (such as atrial fibrillation), lacunar infarction, cryptogenic stroke, and others (such as cervical artery dissection). All imaging and clinical data were generated during the routine clinical workup of the patients in our stroke center. The local ethics committee approved the study.
Brain Imaging Methods MRI was performed during the first 6 hours of symptom onset using a 3-T whole-body MR General Electric
Peripheral venous blood samples were obtained from all patients before the MRI and any treatment. When an ischemic stroke was diagnosed, the samples on days 1 and 7 were repeated. Fresh venous blood was collected in gel-containing Vacutainer tubes (Becton-Dickinson, Plymouth, UK) and then centrifuged at 4500 rpm at 4 C for 10 minutes within 2 hours after withdrawal. Serum was aliquoted into many aliquots and stored at 280 C until analyses.6,7 Lipid Parameters Fresh serum lipids were analyzed using routine methods on a Konelab 30i analyzer (Thermo Electron Corporation, Waltham, Massachusetts). Triglycerides were measured using an automated enzymatic method (Diasys Diagnostic Systems GMBH, Condom, France).8 Total cholesterol and direct high-density lipoprotein (HDL)cholesterol were determined based on recommendations of ARCOL (Comite Franc¸ais de Coordination des Recherches sur l’Atherosclerose et le Cholesterol) using automated enzymatic methods9-11 (Konelab Thermo Fisher Scientifics Cergy-Pontoise, France). LDL-cholesterol was calculated using the Friedewald formula.12 Measurement of Lp-PLA2 Mass and Activity Frozen, collected serum aliquots were thawed at 4 C and assayed for Lp-PLA2 mass and activity. Lp-LA2 mass was assessed using the Plac test enzyme-linked immunosorbent assay kit (Diadexus Inc, South San Francisco, CA) that uses 2 monoclonal antibodies that are
PROGNOSTIC VALUE OF Lp-PLA2 IN ACUTE STROKE 13
specific for Lp-PLA2. Lp-PLA2 activity was performed using an automated colorimetric method on a Konelab 30i analyzer using a rate reaction assay with 1myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine as a substrate (Diadexus Inc; Eurobio, France). The longterm stability of Lp-PLA2 has been demonstrated in frozen serum samples. Activity was expressed as nanomoles of platelet-activating factor hydrolyzed per minute per milliliter of serum (nmoL/min/mL). All assays were performed in the Department of Metabolic Biochemistry of Pitie-Salp^etriere Charles Foix Hospital.
Data Analysis and Statistical Methods Descriptive data are presented as the mean 6 standard deviation or median and interquartile range (IQR). The distributions of Lp-PLA2 mass and activity were normal. Proportions were compared using the chi-squared test. One-way analysis of variance (ANOVA) with the diagnosis (ischemic strokes versus ICH) as a between-subject factor and the LDL-level and the level of atheroma as covariates was used to compare the Lp-PLA2 mass and activities. Atheroma severity was scored by a trained cardiologist as follows: 0 for no atheroma at all, 1 for plaques, 2 for stenosis under 70% ECST (European Carotid Surgery Trial) or 60% NASCET (North American Symptomatic Carotid Endarterectomy Trial), and 3 for more severe stenosis. The temporal evolution of Lp-PLA2 levels in ischemic strokes was investigated using repeated-measures ANOVA with the Lp-PLA2 mass or activity as the dependent variable and the time (days 0, 1, and 7) as the within-subject factor. Post hoc t tests were then performed. Correlations were computed using the bilateral Pearson correlation coefficients (MedCalc Package, version 9.3.2.0, Belgium).
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score at day 1 or 7. There was no significant correlation with the 3-month outcome assessed using the modified Rankin scale score. The initial infarct volume or final infarct volume did not correlate with either the mass or activity. However, Lp-PLA2 mass correlated with activity (r 5 .380, 95% CI: .344-.655, P 5 .005). The temporal evolution analysis (Fig 2) indicated that Lp-PLA2 activity remained stable over time (P 5 .60) whereas Lp-PLA2 mass decreased (F(1,60): 4.06, P 5 .021). Post hoc t tests revealed that Lp-PLA2 mass was lower at day 7 compared with day 1 (median at day 1: 285 mg/L, IQR: 232-323 versus median at day 7: 257, IQR: 209-320; P 5 .007). LDL-cholesterol also decreased between days 1 and 7 (median at day 1: 1.14 g/L, IQR: .78-1.47 versus median at day 7: .90 g/L, IQR: .70-1.20; P 5 .0002). Statins were provided to 30% of the included population before admission, 37% (20 of 54) at day 1 and 61% (33 of 54) at day 7. We finally divided our population according to the etiology of stroke (atherosclerotic n 5 15, cardiogenic n 5 18, lacunar n 5 2, cryptogenic n 5 11, and others n 5 8), and we have examined the differences in both Lp-PLA2 mass and activity between atherosclerotic infarction and other etiologies. No differences between the subgroups were found (Supplementary figure 1). For ICH, Lp-PLA2 activity measured during the first 6 hours of stroke onset correlated with hematoma volume at admission (r 5 .740, 95% CI: .320-.917, P 5 .003) and at follow-up (r 5 .731, 95% CI: .303-.914, P 5.004; Fig 3). LpPLA2 mass did not correlate with the volume at admission and follow-up. No significant correlation was found for either Lp-PLA2 mass and activity with clinical score during the length of stay or for the 3-month outcome.
Discussion Results Sixty-eight patients were recruited prospectively after sudden-onset focal deficits. The diagnoses were ischemic stroke (79%, n 5 54) and ICH (21%, n 5 14). The median time delay from stroke onset to brain imaging was 3.3 hours (IQR: 1.9-4.2 hours). Table 1 shows the clinical and biological characteristics of the entire population and subgroups.
Comparison of Lp-PLA2 Levels Between Ischemic versus ICH The ANOVA test revealed a global overall significant difference in Lp-PLA2 mass based on the diagnosis (F(1,64): 10.7, P 5 .002) but not for the Lp-PLA2 activity (P 5 .38). Post hoc analyses revealed that Lp-PLA2 mass was higher in hemorrhagic than in ischemic stroke (Fig 1).
Prognostic Value in Stroke Patients For ischemic stroke patients, Lp-PLA2 mass and activity at admission (,6 hours of stroke onset) did not correlate with the NIHSS score at admission or the NIHSS
In our study, we observed that Lp-PLA2 mass differed between stroke diagnoses (ICH versus ischemic strokes). Moreover, Lp-PLA2 activity (,6 hours) correlated with hematoma volumes at admission and follow-up. Lp-PLA2 levels were not associated with greater clinical severity or larger ischemic stroke volumes as judged using brain imaging. Finally, in ischemic stroke, the Lp-PLA2 mass decreased during the first week when patients were provided with statins,6 whereas its activity remained stable. Lower Lp-PLA2 mass in ischemic compared with hemorrhagic strokes was a surprising finding. The first reason is that previous studies reported a higher risk of spontaneous hemorrhagic stroke in persons with low cholesterol levels.14,15 Therefore, we theorize that lower Lp-PLA2 levels may also characterize this ICH subpopulation. Second, Lp-PLA2 has been implicated in atherosclerotic processing and should thus be more involved in the physiopathology of ischemic stroke. Indeed, the presence of oxidized phospholipids and increased Lp-PLA2 has been also demonstrated in experimental nonatherosclerotic cerebral ischemia.16,17 It should be also
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Table 1. Descriptive characteristics Clinical and biological characteristics Age (y) Gender % (n) Admission NIHSS 3-month mRS 0-1% (n) Lp-PLA2 mass (mg/L) Lp-PLA2 activity (nmol/min/ml) LDL level (g/L) HDL level (g/L) Total cholesterol level (g/L) Triglycerides (g/L) Known hypertension % (n) Known diabetes % (n) Statins at admission % (n) Tobacco use % (n)
All (n 5 68)
Ischemic stroke (n 5 54)
Hemorrhagic stroke (n 5 14)
P value
70 55-81 56% (38) 13 6-19 28% (19) 307 249-360 163 130-194 1.13 .76-1.40 .51 .41-.67 1.91 1.52-2.25 1.05 .86-1.36 66% (45) 18% (12) 28% (19) 18% (12)
70 55-83 46% (25) 10 3-17 31% (17) 291 248-342 160 124-186 1.15 .85-1.41 .51 .41-.68 1.9 1.50-2.30 1.10 .90-1.30 65% (35) 17% (9) 30% (16) 19% (10)
73 55-81 92% (13) 16 11-20 14% (2) 406 290-430 183 152-211 0.93 .71-1.28 .53 .42-.64 1.7 1.50-2.10 .90 .70-1.80 71% (10) 21% (3) 21% (3) 14% (2)
.98 .005 .054 .35 .001 .10 .20 .72 .29 .38 .91 .96 .73 .96
Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; Lp-PLA2, lipoprotein-associated phospholipase A2; mRS, modified Rankin scale; NIHSS, National Institutes of Health Stroke Scale. Data are shown as the median and interquartile range.
noticed that the initial blood sample analyzed in our cohort was taken in the first 6 hours of stroke onset. LpPLA2 is thought to have a half-life of about a few days, related to the LDL particles half-life. This rather long period is not compatible with an acute increase of LpPLA2. However, the exact half-life of Lp-PLA2 is not known yet. Because Lp-PLA2 is considered a specific marker of vascular inflammation, it may also be elevated in ICH, for example, as a marker of hypertensive vasculopathy. This finding is consistent with the study of Romero et al.18 on APOε2- or ε4-allele carrying patients. In that study, Lp-PLA2 levels were associated with the deep loca-
tion of microbleeds, which is typical of a pattern of hypertensive angiopathy. It is possible that Lp-PLA2 itself plays a role in the rupture of vessel-wall integrity via inflammation processes.19,20 Indeed, a reason that might explain the increase in Lp-PLA2 in ICH patients could be secondary changes of the brain tissue around the hematoma. Actually, as minutes after onset of ICH, the extravasated blood components and the associated molecular patterns released from necrotic and damaged tissue impose a strong proinflammatory state in the adjacent viable brain cells. Therefore, it may be possible that the increase in LpPLA2 in ICH may be because of the infiltration of white cells with a proinflammatory phenotype around the
Figure 1. Lp-PLA2 activity (A) and mass (B) measured at admission for intracranial cerebral hemorrhages (ICH) and ischemic strokes (IS). Lp-PLA2, lipoprotein-associated phospholipase A2.
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Figure 2. Temporal evolution of Lp-PLA2 mass, activity, and LDL-cholesterol level in the ischemic stroke patients from admission to day 7. LDL, low-density lipoprotein; LpPLA2, lipoprotein-associated phospholipase A2. *p ,.05
hematoma.21,22 The same hypothesis could also explain the correlation between Lp-PLA2 and the size of the hematoma. However, the mechanisms underlying this association remain unknown and must be specified. We did not find any correlation between clinical stroke severity and Lp-PLA2 levels (mass or activity) during the first week of stroke. These results are consistent with those of Delgado et al.,3 where no correlation between baseline clinical severity, as assessed using NIHSS score, and Lp-PLA2 was reported in a population of rt-Pa-treated patients. Moreover, in the PROVE-IT (Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis In Myocardial Infarction) trial,23 the baseline Lp-PLA2 measured immediately after the coronary event did not correlate with the risk of recurrence whereas the measurements after 1 month did. Finally, the main reason why Lp-PLA2 mass or activity did not differ between stroke etiology groups could be the relatively small size of our population. However, Lp-PLA2 mass and activity were high in our population, in comparison with controls. In healthy controls,6 mean Lp-PLA2 mass level was 163 6 43 mg/L (166 6 45 mg/L in men and 159 6 39 mg/L in women). For ICH, Lp-PLA2 was a biological marker of severity during the acute stage. In particular, Lp-PLA2 activity
Figure 3. Regression analysis. Plot of the initial (A) and follow-up ICH volume (B) as a function of the Lp-PLA2 activity at admission. A higher Lp-PLA2 activity at admission indicates a greater ICH volume. Lp-PLA2, lipoprotein-associated phospholipase A2.
correlated with hematoma volume at admission and at follow-up: higher Lp-PLA2 activity indicated a larger volume for the intracerebral hemorrhage. Because hematoma volume is a surrogate of the clinical outcome in ICH24 and correlates with Lp-PLA2, Lp-PLA2 may also be predictive of the outcome. Our small sample size and our rather short follow-up may underlie the fact that we did not observe this finding in our study. Moreover, the correlation was mainly driven by the patients with bigger volumes of ICH. However, when removing the patient with the biggest ICH volume, the correlation between Lp-PLA2 activity at admission and hematoma volume at day 1 was still significant (correlation coefficient: .614, P 5 .02). These preliminary findings must be confirmed in a larger cohort to determine whether they can be used in a clinical setting. This study suffers from limitations. Because our primary hypothesis was that Lp-PLA2 was significantly higher and meaningful in ischemic stroke, we did not plan assessments of temporal evolution in the ICH group. It would be interesting to study Lp-PLA2 levels in this latter group to determine whether the levels decrease with the resorption of the hematoma. Secondly, our sample size was too small to enable subgroup analyses, for example, between lobar and deep hemorrhages, or to
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compare different ICH etiologies. Moreover, whether increased Lp-PLA2 is causal or secondary to ICH needs to be determined by basic science experimentations and prospective clinical trials. However, such a thing has never been signaled in all the previous studies on LpPLA2 in primary or secondary prevention and needs further investigations.
Conclusions Our main result is that LpPLA2 mass is higher in ICH than in ischemic stroke. Moreover, we showed that LpPLA2 activity, as measured during the first 6 hours of stroke onset, correlated with hematoma volume at follow-up and could serve as prognostic markers. This suggests that LpPLA2 could provide different information at the hyperacute stage of stroke, especially in ICH. More studies on Lp-PLA2 mass and activity are required in brain hemorrhages to detail and confirm our results. Acknowledgments: The authors thank Franc¸oise Toublan for her valuable assistance with the organization of this study. The Lp-PLA2 kits were provided by DiaDexus. Conflicts of interest: Although Lp-PLA2 measurement kits were provided by Diadexus, at no cost, Diadexus played no role in the study design or statistical analyses. David Rosenbaum has received speaking fees from Diadexus.
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Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013. 11.024.
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