Garcia et al. Journal of Translational Medicine (2023) 21:131 Journal of https://doi.org/10.1186/s12967-022-03867-w Translational Medicine RESEARCH Open Access Soluble low‑density lipoprotein receptor‑related protein 1 as a surrogate marker of carotid plaque inflammation assessed by 18F‑FDG PET in patients with a recent ischemic stroke Eduardo Garcia1,2†, Pol Camps‑Renom3†, Núria Puig4,5, Alejandro Fernández‑Leon6, Ana Aguilera‑Simón3,5, Aleyda Benitez‑Amaro1, Arnau Solé3, David Vilades7,8, José Luis Sanchez‑Quesada4,9, Joan Martí‑Fàbregas3, Elena Jiménez‑Xarrié3, Sonia Benitez4,9*†   and Vicenta Llorente‑Cortés1,8*†    Abstract Background 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) identifies carotid plaque inflam‑ mation and predicts stroke recurrence. Aim Our aim was to evaluate the performance of soluble low-density lipoprotein receptor-related protein 1 (sLRP1) as an indicator of carotid plaque inflammation. Methods A prospective study was conducted among adult patients with recent (< 7 days) anterior circulation ischemic stroke and at least one atherosclerotic plaque in the ipsilateral internal carotid artery. Patients underwent an early (< 15 days from inclusion) 18F-FDG PET, and the maximum standardized uptake value (SUVmax) within the plaque was measured. sLRP1 levels were measured in plasma samples by ELISA. The association of sLRP1 with SUV‑ max was assessed using bivariate and multivariable linear regression analyses. Hazard ratios (HR) were estimated with Cox regression to evaluate the association between circulating sLRP1 and stroke recurrence. Results The study was conducted with 64 participants, of which 57.8% had ≥ 50% carotid stenosis. The multivariable linear and logistic regression analyses showed that sLRP1 was independently associated with (i) SUVmax within the plaque (β = 0.159, 95% CI 0.062–0.257, p = 0.002) and (ii) a probability of presenting SUVmax ≥ 2.85 g/mL (OR = 1.31, 95% CI 1.00–1.01, p = 0.046), respectively. Participants with stroke recurrence showed higher sLRP1 levels at baseline [6447 ng/mL (4897–11163) vs. 3713 ng/mL (2793–4730); p = 0.018]. † Eduardo Garcia and Pol Camps-Renom are contributed equally to this work † Sonia Benitez and Vicenta Llorente-Cortés are contributed equally to this work *Correspondence: Sonia Benitez sbenitez@santpau.cat Vicenta Llorente‑Cortés vicenta.llorente@iibb.csic.es; cllorente@santpau.cat Full list of author information is available at the end of the article © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 2 of 10 Conclusions sLRP1 was independently associated with carotid plaque inflammation as measured by 18F-FDG PET in patients with recent ischemic stroke and carotid atherosclerosis. Keywords Soluble LRP1, 18F-FDG PET, Carotid atherosclerosis, Ischemic stroke recurrence, Vascular inflammation Introduction mediated by several metalloproteinases [13, 14]. LRP1 Cardiovascular disease is a main cause of death and dis- shedding can be induced by atherogenic lipoproteins in ability worldwide [1]. Atherosclerosis from the internal human coronary vascular smooth muscle cells [15], by carotid artery is responsible for 15–20% of all ischemic tissue plasminogen activator (tPA) in astrocytes [16], strokes [2]. Patients with recent symptomatic carotid and by inflammatory mediators in macrophages [17]. In plaques show a three-fold risk of early stroke recurrence the context of stroke, sLRP1 seem to play a neuropro- as compared to other stroke subtypes [3]. In addition, tective role in hemorrhagic stroke by removing heme- stroke survivors are at risk of coronary events or recur- hemopexin complexes, but it may also induce deleterious rent strokes despite guideline-based treatments [4–6]. effects in ischemia by eliciting blood–brain barrier per- Therefore, there is a need for new diagnostic biomark- meability and glial inflammation [18]. ers to help assess the residual risk prediction of vascular The soluble LRP1 form seems to have functions that events after an atherothrombotic stroke. are independent of those of the cellular LRP1 form [18]. Inflammation has emerged as a key feature of carotid The cellular form is involved in the regulation of vascu- plaque vulnerability. Of note, it can be measured non- lar integrity [18, 19] and vascular lipoprotein metabo- invasively by 18F-fluorodeoxyglucose positron emission lism [20–22]. In particular, cellular LRP1 receptor plays tomography (18F-FDG PET) [7]. A recent study revealed a crucial role in the generation of foam cells, which 18 become proinflammatory cells [22, 23] with high capacity F-FDG PET to be a mainstay technique for stroke prog- nosis, based on an independent association between to release sLRP1 [15, 17, 22]. The role of sLRP1 is rather maximal standardized uptake value (SUVmax) within unknown and could be, at least partially determined the plaque and early stroke recurrence in patients with by the competition with membrane LRP1 for certain carotid stenosis [8, 9]. Interestingly, a SUVmax threshold ligands. of 2.85 g/mL discriminates stroke recurrence with high On the basis of these previous results, the aims of this sensitivity, even in patients with moderate stenosis. How- work were to determine: (i) the performance of sLRP1 as ever, some intrinsic limitations of this imaging technique, an indicator of carotid plaque inflammation evaluated by 18 such as its high cost and limited availability, may hinder F-FDG PET and (ii) the value of sLRP1 as a prognostic its implementation in clinical practice. biomarker for stroke recurrence. In this regard, finding plasma biomarkers associated with plaque inflammation assessed by 18F-FDG PET could be of great interest, as they would eventually serve Methods Study participants and study design as screening tools as well as markers for monitoring An observational cohort study (NCT03218527) was response to preventive treatments. Recent reports show conducted with consecutive adult patients with a recent that circulating concentrations of fractalkine (FKN), sol- anterior circulation ischemic stroke and carotid athero- uble vascular cell adhesion molecule 1 (sVCAM-1), and sclerosis. The study was conducted in our center between soluble intercellular cell adhesion molecule 1 (sICAM-1) January 2016 and March 2019. It was approved by the are associated positively with higher degrees of plaque Ethics Committee of Hospital Santa Creu i Sant Pau inflammation; in particular, sICAM-1 predicts with high (IIBSP-LRB-2017-54), and patients gave written consent sensitivity the risk of finding highly inflamed carotid for participation. The study was performed in accordance plaques and the risk of stroke recurrence within one year with the Helsinki Declaration. [10]. Participants of the current study were the same who Soluble low-density lipoprotein receptor-related pro- had been included in a previous publication [10]. Briefly, tein 1 (sLRP1) has been recently postulated to be a pre- patients were required to fulfill the following inclu- dictive biomarker for coronary artery disease (CAD) sion criteria: (1) the age limit was fixed in ≥ 50 years to risk in a case-cohort study based on the follow-up of the minimize the inclusion of non-atherosclerotic vasculopa- REGICOR population-based cohort, in which no par- thies; (2) anterior circulation ischemic stroke or transient ticipants had a previous history of cardiovascular dis- ischemic attack (TIA) within the seven days preceding ease [11]. sLRP1 is generated by constitutive or induced the study inclusion; (3) at least one atherosclerotic plaque intramembrane proteolysis of the cell receptor [12] in the internal carotid artery (ICA) on the side consistent Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 3 of 10 with stroke symptoms, regardless of the degree of steno- 5 proximal). 18F-FDG was quantified using standardized sis [24]; and (4) a modified Rankin Scale (mRS) score < 4 uptake values [SUV g/mL, defined as measured uptake before inclusion. Exclusion criteria were: (1) the presence (MBq/mL)/injected dose (MBq) per patient weight (g)]. of definitive cardioembolic, lacunar, or unusual stroke The single hottest slice was defined as the axial slice with etiology according to the TOAST criteria [25]; (2) hemo- maximal SUV uptake (SUVmax) [7]. dynamic stroke/TIA; (3) prior carotid surgery or stenting; (4) life-expectancy < 1 year; or (5) suspicion of concomi- Plasma and serum determinations tant infections at the time of blood extraction. Peripheral blood samples from the participants were The following variables related to vascular risk and collected at day 7 ± 1 after the stroke/TIA. Plasma was stroke characteristics were recorded for all participants: collected in ethylenediaminetetraacetic acid (EDTA)- (1) age and sex; (2) medical history, including hyperten- containing Vacutainers and serum was collected in sion, diabetes, dyslipidemia, prior stroke/TIA, coronary Serum Separator Tubes coated with clot activator. Tubes artery disease, smoking, and alcohol abuse; (3) previous were centrifuged at 1500g for 15 min at 4 ℃, and aliquots treatments; (4) National Institutes of Health Stroke Scale were frozen at – 80 ℃ until analysis. (NIHSS) score at admission; (5) body mass index (BMI); Serum lipid profile (triglycerides, total cholesterol, low- (6) adherence to regular physical exercise, according to density lipoprotein cholesterol (LDLc), and high-density the physician-based assessment and counseling for exer- lipoprotein cholesterol (HDLc), creatinine, hemoglobin, cise (PACE) scale [26]; (7) Mediterranean diet adherence and high sensitivity CRP (hsCRP) were measured in an according to the PREDIMED score [27]; (8) mRS score autoanalyzer (Alinity ci-series, Abbott Core Laboratory, at inclusion; (9) stroke etiology according to the TOAST Chicago, Illinois, USA). Glycated hemoglobin (Hba1c) criteria after a diagnostic work-up that included a 24-h was quantified using high-performance liquid chroma- (or longer) electrocardiogram monitoring, an echocardi- tography in total blood. ogram, and an ultrasound carotid examination; and (10) results from the admission blood test, including renal function, blood cell counts, hemostasis, and lipid profile. Measurement sLRP1 concentrations sLRP1 concentrations were measured in frozen plasma Atherosclerosis imaging by 18F‑FDG PET and assessment using commercially available enzyme-linked immuno- of plaque inflammation in the internal carotid artery sorbent assay (ELISA) (Uscn Life Science Inc. China) All stroke patients underwent a baseline medical assess- according to the manufacturer’s recommendations. The ment by trained study personnel and a carotid 18F-FDG assay had a within- and between-assay coefficient of vari- PET/CT study in less than 15 days from the inclusion. ation lower than 10%, and 12%, respectively, and a limit The blood sample was collected at day 7 ± 1 from the of detection of 0.156 ng/mL. The assay showed no signifi- index stroke. The treating clinicians provided medical cant cross-reactivity or interference between LRP1 and and revascularization treatments according to current analogues. guidelines [28]. Carotid 18F-FDG PET was performed in a Philips Follow‑up and outcomes Gemini TF TOF 64 PET/CT (Philips Medical System, Standardized follow-up assessments were performed Eindhoven, and Netherlands) as previously described for all participants on day 90 from the index stroke, and [8, 9]. The examinations were performed after a fast of every 6 months thereafter until the study terminated. at least six hours. PET scans were not performed if pre- The primary outcome was to determine the asso- PET blood glucose levels exceeded 10 mmol/L. Two ciation between clinical, imaging, and laboratory vari- hours before image acquisition, 320 MBq of 18F-FDG ables (including sLRP1 levels) collected in the study and was administered. The uptake phase was standardized carotid plaque inflammation assessed by 18F-FDG PET/ with the patient resting. PET images were acquired in CT, as previously described. 3-dimensional mode in 2-bed positions for 10 min each. The secondary outcome was any anterior circulation CT angiography was usually done at admission, some- ipsilateral recurrent stroke along patient follow-up. times before PET/CT. CT angiography and PET images A recurrent stroke was defined as a new sudden onset were co-registered afterwards to assess the slice of maxi- of persistent (≥ 24 h) neurologic deficit after the index mal plaque stenosis. event, or a new persistent or transient neurologic defi- 18 F-FDG activity was measured in 10 regions of inter- cit with imaging confirmation of a new cerebral infarc- est, which were defined relative to the slice of maxi- tion. Only strokes unrelated to a revascularization mal stenosis on the co-registered CT angiography, procedure (defined as any event > 24 h after carotid corresponding to a 1 mm axial plaque slice (5 distal and revascularization) were considered outcomes. During Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 4 of 10 the follow-up, nine stroke recurrences were registered Results in our population that represent a risk of 14.1%. Of Clinical characteristics of the study population and sLRP1 them, five recurrences occurred before PET (7.8%) and levels four recurrences occurred after PET (6.3%). Clinical and biochemical parameters for the studied population are summarized in Additional file 1: Table S1. The study included 64 patients, 37 (57.8%) with a ≥ 50% Statistical analyses carotid stenosis, and the remainder with mild stenosis Data were presented as mean and standard deviation (< 50%). Within the participant cohort, the average age (SD) for continuous variables, median and interquar- was 74.9 ± 9.6 years, 75% were men, 82% had hyperten- tile range for continuous variables not normally dis- sion, 42% had type 2 diabetes mellitus, 59.3% had obesity, tributed, and frequencies (percentages) for categorical and 67.2% were current smokers. variables. Data normality was analyzed using the Kol- The SUVmax was significantly higher in patients with mogorov–Smirnov test. Continuous variables were more than 50% stenosis [2.74 g/mL (2.41–3.23) vs. 2.39 g/ compared between groups using the Student’s t-test or mL (2.23–2.69); p = 0.034], whereas there were no signifi- the Wilcoxon Rank Sum test (when a non-parametric cant differences in the rest of clinical variables dichoto- test was required). Categorical variable comparisons mizing the participants according to stenosis degree (e.g., were performed using the chi-square test. The Spear- higher or lower than 50%). We found that sLRP1 levels man’s test was performed to evaluate the correlations were not different according to the degree of stenosis between sLRP1 and other variables. The association [stenosis ≥ 50%: 3915 ng/mL (3190–4730) vs. stenosis between circulating sLRP1 levels and plaque inflam- < 50%: 3596.7 ng/mL (2665–5130); p = 0.688]. mation assessed by SUVmax in the 18F-FDG PET/CT Spearman’s test showed that sLRP1 positively corre- was analyzed. After a bivariate analysis including sLRP1 lated with triglycerides (­ r2 = 0.426, p < 0.001) and abdom- levels and all the variables collected in the study, we inal perimeter ­ (r2 = 0.304, p < 0.024). In addition, the performed a multivariable analysis using linear regres- correlation of sLRP1 with some inflammatory molecules sion. From a maximal model including all the variables including sICAM1, sVCAM1 and FKN (found associated showing a trend towards significance in the bivariate with plaque inflammation in the same cohort of patients) analysis (p < 0.1), a backward stepwise selection mod- [9] was analyzed. In particular, sLRP1 positively cor- eling was performed, and only variables with a signifi- related with sICAM-1 (p = 0.009) and showed a strong cance level of p < 0.05 were kept in each iteration. Data trend towards significance with sVCAM-1 (p = 0.087) were expressed as beta coefficient with its 95% CI and and FKN (p = 0.066) (Table 1). standardized beta (β). The following variables were log-transformed to achieve a normal distribution when Circulating sLRP1 concentrations are associated constructing the linear regression models: SUVmax, with carotid plaque inflammation sLRP1, triglycerides, LDLc, HDLc, and high sensitivity Carotid plaque inflammation was assessed by 18F-FDG CRP. PET imaging in accordance with the study protocol for The association of circulating sLRP1 and the prob- 53 of 64 participants (24 in the < 50% stenosis group, and ability of presenting an intraplaque SUVmax ≥ 2.85 g/ 29 in the ≥ 50% stenosis group). In three cases, although 18 mL was also studied. Bivariate and multivariable logis- F-FDG PET imaging was performed, the carotid tic regression analyses were performed using the same variables selected in the multivariable linear regression analysis. The results are presented as odds ratio (OR) Table 1 Analysis of sLRP1 association with inflammatory and and 95% confidence intervals (CI). ROC analyses were metabolic variables performed to compare the predictive value of sLRP1 Correlation coefficient p and high-sensitivity C-reactive protein (hsCRP) to identify highly inflamed plaques. sICAM-1 0.337 0.009 Finally, a survival analysis was conducted using Cox sVCAM-1 0.225 0.087 regression to test the association between sLRP1 levels FKN 0.239 0.066 and stroke recurrence during the follow-up. TG 0.426 < 0.001 The statistical software package SPSS 15.0 for Win- Abdominal perimeter 0.304 < 0.024 dows (SPSS Inc., Chicago, IL, USA) and Stata v.15 The correlation coefficient and p-value were obtained by Spearman correlation (Texas, US) were used for the statistical analyses. Dif- test ferences were considered statistically significant when sLRP1 soluble low-density lipoprotein receptor-related protein 1, sICAM-1 soluble intercellular adhesion molecule-1, sVCAM-1 soluble vascular adhesion p < 0.05. molecule-1, FKN fractalkine, TG triglycerides Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 5 of 10 inflammation was not assessable due to acquisition prob- 2.85 g/mL showed increased sLRP1 levels in patients lems. In eight cases, 18F-FDG PET was not performed. In with SUVmax ≥ 2.85 g/ml as compared to those with four cases, the patients underwent carotid revasculariza- SUVmax < 2.85 g/mL [5130 ng/mL (3496.7–7430) vs. tion before PET examination, and the other four patients 3846.65 ng/mL (2630–4730); p = 0.032] (Fig. 1). refused to undergo PET. The multivariable linear regression analysis showed We found a positive association between SUVmax and an independent association with SUVmax for BMI the following clinical variables: BMI (β = 0.008, 95% CI (β = 0.006, 95% CI 0.000–0.0013, p = 0.041), the 0.001–0.015, p < 0.016), waist circumference (β = 0.003, degree of carotid stenosis (β = 0.041, 95% CI 0.000– 95% CI 0.001–0.005, p = 0.004), and PACE (β = − 0.015, 0.082, p = 0.047), and much strongly for sLRP1 levels 95% CI − 0.028–0.002, p = 0.023) (Additional file 1: (β = 0.159, 95% CI 0.062–0.257, p = 0.002) (Table 2). Table S2), according to our previous study [9]. In addition, the multivariable logistic regression anal- The strongest association was observed for sLRP1, ysis models showed that, after adjusting for BMI and which was also significantly and positively associated degree of carotid stenosis, only circulating sLRP1 lev- with carotid plaque inflammation (β = 0.192, 95% CI els predicted highly inflamed carotid plaques (SUV- 0.094–0.290, p < 0.001) (Additional file 1: Table S2). max ≥ 2.85 g/mL) (OR = 1.31, 95% CI 1.00–1.01, There were not differences in sLRP1 levels between p = 0.046) (Additional file 1: Table S3). patients with (n = 35) or without (n = 29) previous treat- A receiver-operator curve (ROC) analysis was per- ment with statins (p = 0.364). Spearman correlation anal- formed to evaluate the sensitivity and specificity of ysis indicated that sLRP1 correlated with SUVmax only sLRP1 for predicting the presence of highly inflamed in the group previously treated with statins (coefficient carotid plaques (SUVmax ≥ 2.85 g/mL). This analy- correlation = 0.563; p = 0.001). sis showed that the optimal sLRP1 cut-off point was The ROC sensitivity/specificity analysis from our 3500 ng/mL. This sLRP1 value predicted the probability previous study, [7] led a balanced cut-off point for pre- for the presence of highly inflamed plaques with a sen- dicting stroke recurrence of 2.85 g/mL. The dichoto- sitivity of 80% and a specificity of 42.11%. Compared mization of SUVmax value by the cut-off point of to hsCRP [area under the curve (AUC) = 0.69 vs. 0.48, p = 0.093], the presence of sLRP1 presented a much higher predictive value for highly inflamed plaques (Fig. 2). sLRP1 was associated with stroke recurrence At follow-up after a median of 10 (4–14) months, a total of 9 stroke recurrences were recorded (with stroke occurring in 5 participants before the PET examination, and in 4, after). While the sLRP1 levels in participants with stroke recurrence was higher, this difference was not significant [4430 ng/mL (2630–7430) vs. 3830 ng/ SUV max < 2.85 g/mL SUV max > 2.85 g/mL mL (2840–4730), p = 0.308]. Of note, when only partic- Fig. 1 Circulating sLRP1 levels in patients with low (SUVmax < 2.85 g/ mL) or high (SUVmax > 2.85 g/mL) carotid plaque inflammation. ipants with stroke recurrence post-PET during the fol- Patients were dichotomized according to SUVmax values by low-up were considered, sLRP1 levels were significantly the cut-off point of 2.85 g/mL, and sLRP1 levels were measured higher at baseline in participants with stroke recur- in plasma-EDTA blood samples by ELISA. Results are shown as rence than in those without recurrence [6447 ng/mL mean ± SD. sLRP1, soluble low-density lipoprotein receptor-related (4897–11,163) vs. 3713 ng/mL (2793–4730); p = 0.018]. protein 1 In a Cox regression analysis adjusted for carotid artery Table 2 Multivariable linear regression analysis of the predictors of carotid plaque inflammation (SUVmax) β-coefficient 95% CI β-standardized p logLRP1 0.1592263 0.0618788–0.2565738 0.3989622 0.002 BMI 0.0064457 0.0002674–0.012624 0.2557793 0.041 Stenosis < or ≥ 50% 0.0413429 0.0005251–0.0821606 0.2405786 0.047 LogLRP1 logarithm of sLRP1, BMI body mass index Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 6 of 10 Supporting the relationship between sLRP1 con- centrations and inflamed carotid plaques, we found a positive correlation between sLRP1 and sICAM-1 lev- els (with near-significance for sVCAM-1 and FKN). sICAM-1, sVCAM-1, and FKN have been previously associated with carotid plaque inflammation; in par- ticular, sICAM-1 was shown to be the best candi- date between these three to predict highly inflamed carotid plaques and the recurrence of stroke within 1 year [10]. Interestingly, the combination of sLRP1 levels (≥ 3500 ng/mL) with sICAM-1 (≥ 240 pg/mL) significantly increased the predictive value for highly inflamed plaques as compared to sLRP1 alone (data not shown). Of note, sLRP1 was closely associated Fig. 2 ROC curve analyses of sLRP1 and hsCRP for discrimination of with intraplaque SUVmax but not with stenosis degree, SUVmax > 2.85 g/mL. hsCRP, high sensitivity C reactive protein; sLRP1, soluble low-density lipoprotein receptor-related protein 1 despite the tight association between SUVmax and the degree of stenosis in this group of patients. These results suggest that plaque inflammation is not only linked to stenosis but also that sLRP1 is likely a better stenosis (≥ 70% vs. < 70%), the association between indicator of the composition and features of plaque vul- sLRP1 levels and post-PET recurrence remained sig- nerability than of the plaque size. Along this line, sev- nificant (p = 0.046). eral studies have previously suggested that sLRP1 may Other variables that were associated with post-PET be a biomarker that reflects the extra- and intracellular recurrence were female sex (p = 0.022), having Diabe- composition of LDL-cholesteryl esters in the athero- tes Mellitus (p = 0.016), and having SUVmax (p = 0.008). sclerotic plaques [30–32]. In addition, in previous stud- Unfortunately, the low rate of outcomes prevented us ies performed with patients with hypercholesterolemia from conducting an adjusted multivariable Cox regres- or type 1 diabetes, or the general population, there was sion analysis; thus, our results should be interpreted a strong correlation between circulating sLRP1 and cautiously. LDL cholesterol concentrations [11, 15, 33, 34]. We did not observe this correlation in this group of patients, Discussion likely due to the treatment of the participants in the The present study suggests that sLRP1 is a surrogate study with statins, which efficiently reduces circulating marker of carotid plaque inflammation. Our main find- sLRP1 levels [15]. These results do not discard the pres- ings are that sLRP1 concentrations i) are independently ence of vascular modified forms of LDL and foam cells associated with the degree of plaque inflammation meas- in association with plaque inflammation. Indeed, foam ured by 18F-FGD PET, and ii) predict highly inflamed cells generated by modified LDL uptake have causal plaques with high sensitivity. effects on carotid and coronary plaque inflammation in These results are novel and highly relevant because, experimental models [14, 22, 23] and in humans [35]. although previous studies had already shown that sLRP1 Moreover, foam cells in the human atherosclerotic circulating levels are associated with the presence of plaques, which are derived mainly from smooth muscle carotid plaque stenosis in experimental models of hyper- cells (SMC) and macrophages [36, 37], take up 18F-FDG cholesterolemia [22] and in patients with hypercholester- and contribute to the SUVmax measured by PET [38, olemia, [15] this is the first time that sLRP1 concentration 39]. All these studies suggest that sLRP1 levels in the has been directly and positively associated with carotid participants of the present study are likely associated plaque inflammation assessed by a highly sensitive with a high number of proinflammatory vascular foam method, such as 18F-FDG PET. In the previous study cells that avidly take up 18F-FDG. conducted with patients with hypercholesterolemia, cir- As previously described in the same group of patients culating sLRP1 levels were found to decrease after statin [10], results from the present study show that SUVmax treatment and to increase after statin withdrawal [15]. As is associated with BMI, waist circumference and PACE, all the participants of the current study were intensively in line with the low-grade chronic inflammation ascribed treated with statins, it would be expected that sLRP1 lev- not only to obese, but also to overweight patients [40]. els, as well as their association with inflammation, would LRP1 levels are also positively correlated with triglyc- be much higher in the absence of statin treatment [29]. erides and BMI in agreement with the close association Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 7 of 10 between sLRP1 and epicardial fat extension in type 1 the relationship between phenotypic manifestation of diabetes and general population previously reported [33, coronary and carotid atherosclerosis suggests that know- 34]. ing the state of carotid arteries whenever CAD is sus- Of note, the ROC analysis showed that sLRP1 levels pected has clinical utility [41, 42]. Based on these results, higher than 3,500 ng/mL predicted the presence of highly further studies in large cohorts are warranted to deter- inflamed carotid plaques (SUVmax ≥ 2.85 g/mL) with mine this putative predictive role of sLRP1 as a dual bio- high sensitivity. The cut-off points of sLRP1 (3500 ng/ marker of carotid and coronary atherosclerosis. mL) found in the present study is similar to sLRP1 values The strengths of our study include the strict carotid 3210 ng/mL [2690–8310] that were significantly associ- vascular characterization through 18F-FDG PET, the pro- ated with higher levels of BMI, glucose, or triglycerides, spective follow-up of patients, and the control for poten- a higher REGICOR risk, and an increased incidence of tial covariates. coronary events in our previous case-cohort study [11]. We also observed an association between sLRP1 levels Limitations and the risk of stroke recurrence during the (10-month) Limitations include the limited number of participants follow-up. This association was greater and statistically in the study due to narrow eligibility criteria, as well as significant when considering only post-PET recurrences. the limited number of recurrences due to the relatively In our cohort, PET/CT was performed within 15 days short follow-up. Further studies with increased numbers from inclusion; thus, indirectly, we may assume that of participants and long-term follow-up are required to sLRP1 levels were associated with latter events rather validate the potential sLRP1 diagnostic and prognostic than early recurrences related to an unstable ruptured value in the context of carotid atherosclerosis and stroke. carotid plaque. Unfortunately, the low rate of outcomes prevented us from conducting an adjusted multivariable Conclusions Cox regression analysis; thus, these results should be In summary (Fig. 3), this study provides new evidence interpreted cautiously. that plasma sLRP1 is independently associated with The results of this study, together with a previous study carotid plaque inflammation and with the probability of showing that the sLRP1 concentration is independently presenting SUVmax ≥ 2.85 g/mL. In addition, here, we associated with CAD risk, [11] support the potential of show that sLRP1 levels are higher in patients with stroke sLRP1 as a dual prognostic biomarker of carotid and cor- recurrence during follow-up. Together, these results onary artery events. A recent meta-analysis comparing support the role of sLRP1 in inflammation and vascular 18F-FDG 18F-FDG PLASMA BLOOD 18F-FDG 18F-FDG sLRP1 18F-FDG CYTOKINES (sICAM-1, sVCAM-1, FKN) 18F-FDG LRP1 18F-FDG VSMC ATHEROSCLEROTI PROINFLAMMATORY C PLAQUE MACROPHAGES CYTOKINES SUVmax 18F-FDG PET/CT LDL HIGHLY INFLAMED CAROTID PLAQUE Fig. 3 The progression of atherosclerotic plaque is associated with an increased number of active cells in the arterial intima (macrophages and smooth muscle cells) that avidly take up 18F-FDG (SUVmax). Many of these cells become foam cells by taking up atherogenic lipoproteins retained in the intimal extracellular matrix, acquiring a highly proinflammatory phenotype that release cytokine and sLRP1 to the circulation. The results of the present study show the close relationship between sLRP1 and proinflammatory cytokines (sICAM-1, sVCAM-1 and FKN) in the blood, as well as between circulating sLRP1 levels and intra-plaque inflammation measured by PET in patients with a recent ischemic stroke. 18F-FDG 18F-fluorodeoxyglucose, SUVmax standardized uptake value, sLRP1 soluble low-density lipoprotein receptor-related protein 1, sICAM-1 soluble intercellular cell adhesion molecule 1, sVCAM-1 soluble vascular adhesion molecule 1 (sVCAM-1), FKN fractalkine Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 8 of 10 pathology by revealing that sLRP1 could be a surrogate Author contributions VLl-C, SB, and PC-R conceived the original idea, designed the study, and wrote biomarker of carotid plaque inflammation in patients the manuscript, with support from EG, NP, JLS-Q, JM-F, and EJ-X. EG, NP, AA-S, with recent ischemic stroke. This knowledge paves the AB-A, and AS conducted the experiments. AF-L was the responsible of image way for further research addressing the putative use of acquisition and analysis. JLS-Q, EJ-X, DV, and PC-R contributed to the design of the study and supervised the findings of the work. All authors read and sLRP1, alone or in combination with other plasma bio- approved the final manuscript. markers, for selecting patients eligible for 18F-FDG PET. This biomarker could also be clinically useful for predict- Funding Open Access funding provided thanks to the CRUE-CSIC agreement with ing the risk of ischemic stroke and other cardiovascular Springer Nature. The economic support to develop this project was received diseases as a complementary information to 18F-FDG from Fundació La Marato TV3 (201716.10), from the Instituto de Salud Carlos PET. III (co-financed by the European Regional Development funds, FEDER), FIS PI15/00884 (to JM-F), FIS PI19/00421 (to PC-R and SB) and FIS PI21/01523 (to VLl-C), from Fundación BBVA Ayudas a equipos de investigación 2019 (“Trans‑ lational Molecular Imaging for Detection of Cholesterol Entrapment in the Vas‑ Abbreviations culature with 68 Ga-labeled LRP1-derived Peptides” to VLl-C), from Sociedad BMI Body mass index Española de Arteriosclerosis Grant 2021 (to SB), and from Acadèmia de Cièn‑ CAD Coronary artery disease cies Mèdiques de Catalunya I Balears Grant (to DV). The team is part of CIBER EDTA Ethylenediaminetetraacetic acid 18 18 of Diabetes and Metabolic Diseases (CIBERDEM, CB07/08/0016 to SB and F-FDG  F-fluorodeoxyglucose JLS-Q) and CIBER Enfermedades Cardiovasculares (CIBERCV; CB16/11/00276 to FKN Fractalkine DV and VLl-C) run by the Instituto de Salud Carlos III. AB-A. (FI19/00205) and Hba1c Glycated hemoglobin NP (FI20/00252) are predoctoral fellows granted by the Programme_Contratos HDLc High-density lipoprotein cholesterol predoctorales de formación de investigación en salud_ from the Instituto hsCRP High-sensitive C reactive protein de Salud Carlos III (ISCIII) and co-financed with ERDFs. EG, AB-A and VLl-C are ICA Internal carotid artery members of Redes de investigación (Enfermedades Metabólicas y Cáncer LDLc Low-density lipoprotein cholesterol RED2018-102799-T), a project run by MINECO. VLl-C, AB-A and DV are mem‑ mRS Modified Rankin scale bers of the Quality Research Group 2017 SGR 946 and SB, NP and JLS-Q of the NIHSS National Institutes of Health Stroke Scale 2017-SGR-1149 group from the Generalitat de Catalunya. SB, PC-R, JM-F and PACE Physician-based assessment and counseling for exercise AA-S are members of RICORS-ICTUS (RD21/0006/0006), and FEDER. VLL-C, EG, PET Positron emission tomography NP; SB and JLS-Q are members of the Spanish Atherosclerosis Society Vascular REGICOR Registre Gironi del Cor Biology Group. IR-SANTPAU is a centre of CERCA Programme/Generalitat de sICAM-1 Soluble intercellular adhesion molecule-1 Catalunya. sLRP1 Soluble low-density lipoprotein receptor-related protein 1 SMC Smooth muscle cells Availability of data and materials SUVmax Maximal standardized uptake value The datasets used and/or analyzed during the current study are available from sVCAM-1 Soluble vascular cell adhesion molecule 1 the corresponding author on reasonable request. TC Total cholesterol TG Triglycerides TIA Transient ischemic attack Declarations tPA Tissue plasminogen activator Ethics approval and consent to participate This observational cohort study (NCT03218527) was approved by the Ethics Supplementary Information Committee of Hospital Santa Creu i Sant Pau (IIBSP-LRB-2017-54), and patients The online version contains supplementary material available at https://​doi.​ gave written consent for participation. The study was performed in accord‑ org/​10.​1186/​s12967-​022-​03867-w. ance with the Helsinki Declaration. Additional file 1: Table S1. Clinical characteristics and biochemical Consent for publication parameters of patients dichotomized according to carotid artery stenosis. All authors have read and agreed to the published version of the manuscript. BMI: Body mass index; HDL: High density lipoproteins; hsCRP: high-sen‑ sitivity C-reactive protein; LDL: Low density lipoproteins; NIHSS: National Competing interests institute of Health Stroke Scale; PACE: Physician-based Assessment and The author(s) declared no potential competing interests with respect to the Counseling for Exercise; PREDIMED: PREvención con DIeta MEDiterránea; research, authorship, and/or publication of this article. SUVmax: Maximum standarized Uptake Value; TC: Total cholesterol; TG: Triglycerides. A chi2 test was used to determine the frequencies and the p Author details 1 value of categorical variables and a Mann-Whitney test to determine the p Lipids and Cardiovascular Pathology, Institut d’Investigacions Biomèdiques value for quantitative variables. Table S2. Bivariate linear regression analy‑ de Barcelona (IIBB)-Spanish National Research Council (CSIC), Institut ses of the association between clinical variables and carotid inflamma‑ d’Investigació Biomèdica Sant Pau (IIB SANT PAU), Sant Quintí 77‑79, tion (SUVmax). BMI: Body mass index; CRP: C reactive protein; HDL: High 08041 Barcelona, Spain. 2 Universitat Autònoma de Barcelona, Barcelona, density lipoproteins; LDL: Low density lipoproteins; LogLRP1: logarithm of Spain. 3 Stroke Unit, Department of Neurology, Hospital de la Santa Creu sLRP1; NIHSS: National institute of Health Stroke Scale; PACE: Physician- i Sant Pau, IIB SANT PAU, Barcelona, Spain. 4 Cardiovascular Biochemistry, based Assessment and Counseling for Exercise; PREDIMED: PREvención Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU), Sant Quintí 77‑79, con DIeta MEDiterránea; sLRP1: Soluble low-density lipoprotein receptor- 08041 Barcelona, Spain. 5 Department of Biochemistry and Molecular Biology, related protein 1; TC: Total cholesterol; TG: Triglycerides. A linear regression Faculty of Medicine, Universitat Autònoma de Barcelona (UAB), Building M, was used to determine the b-coefficient, the β- standardized and the p Cerdanyola del Vallés, Barcelona, Spain. 6 Department of Nuclear Medicine, value. Table S3. Logistic regression analyses of the association between Hospital de la Santa Creu i Sant Pau, IIB SANT PAU, Barcelona, Spain. 7 Cardiac sLRP1 and metabolic variables with carotid inflammation in patients with Imaging Unit, Department of Cardiology, Hospital de la Santa Creu i Sant Pau, SUVmax ≥ 2.85 g/ml. sLRP1: Soluble LRP1; Stenosis < or = > 50%: patients IIB SANT PAU, Barcelona, Spain. 8 CIBER of Cardiovascular (CIBERCV), Instituto dichotomized as having 50% or more stenosis or less than 50% stenosis; de Salud Carlos III, Madrid, Spain. 9 CIBER of Diabetes and Metabolic Diseases BMI: Body mass index. A logistic regression was used to determine the (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain. odds-ratio and the p value. Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 9 of 10 Received: 14 October 2022 Accepted: 29 December 2022 18. Bhattacharya A, Ashouri R, Fangman M, Mazur A, Garett T, Doré S. Soluble receptors affecting stroke outcomes: potential biomarkers and therapeu‑ tic tools. Int J Mol Sci. 2021;22(3):1108. 19. Lillis AP, Van Duyn LB, Murphy-Ullrich JE, Strickland DK. LDL receptor- related protein 1: unique tissue-specific functions revealed by selective References gene knockout studies. Physiol Rev. 2008;88(3):887–918. 1. Li Z, Lin L, Wu H, Yan L, Wang H, Yang H, et al. Global, regional, and 20. Llorente-Cortés V, Otero-Viñas M, Sánchez S, Rodríguez C, Badimon L. national death, and disability-adjusted life-years (DALYs) for cardiovascu‑ Low-density lipoprotein upregulates low-density lipoprotein receptor- lar disease in 2017 and trends and risk analysis from 1990 to 2017 using related protein expression in vascular smooth muscle cells: possible the global burden of disease study and implications for prevention. Front involvement of sterol regulatory element binding protein-2-dependent Public Health. 2021;9:559751. mechanism. Circulation. 2002;106(24):3104–10. 2. Chaturvedi S, Bruno A, Feasby T, Holloway R, Benavente O, Cohen SN, 21. Costales P, Fuentes-Prior P, Castellano J, Revuelta-Lopez E, Corral-Rod‑ et al. Carotid endarterectomy–an evidence-based review: report of the ríguez MÁ, Nasarre L, et al. KDomain CR9 of low density lipoprotein (LDL) Therapeutics and Technology Assessment Subcommittee of the Ameri‑ receptor-related protein 1 (LRP1) Is critical for aggregated LDL-induced can Academy of Neurology. Neurology. 2005;65(6):794–801. foam cell formation from human vascular smooth muscle cells. J Biol 3. Lovett JK, Coull AJ, Rothwell PM. Early risk of recurrence by subtype Chem. 2015;290(24):14852–65. of ischemic stroke in population-based incidence studies. Neurology. 22. Bornachea O, Benitez-Amaro A, Vea A, Nasarre L, de Gonzalo-Calvo D, 2004;62(4):569–73. Escola-Gil JC, et al. Immunization with the Gly1127–Cys1140 amino acid 4. Mohan KM, Wolfe CD, Rudd AG, Heuschmann PU, Kolominsky-Rabas sequence of the LRP1 receptor reduces atherosclerosis in rabbits Molecu‑ PL, Grieve AP. Risk and cumulative risk of stroke recurrence: a systematic lar, immunohistochemical and nuclear imaging studies. Theranostics. review and meta-analysis. Stroke. 2011;42(5):1489–94. 2020;10(7):3263–80. 5. Touzé E, Varenne O, Chatellier G, Peyrard S, Rothwell PM, Mas JL. Risk of 23. Bäck M, Yurdagul A Jr, Tabas I, Öörni K, Kovanen PT. Inflammation and its myocardial infarction and vascular death after transient ischemic attack resolution in atherosclerosis: mediators and therapeutic opportunities. and ischemic stroke: a systematic review and meta-analysis. Stroke. Nat Rev Cardiol. 2019;16(7):389–406. 2005;36(12):2748–55. 24. Bartlett ES, Walters TD, Symons SP, Fox AJ. Quantification of carotid steno‑ 6. Climent E, Benaiges D, Pedro-Botet J. Lipid-lowering treatment in sis on CT angiography. AJNR Am J Neuroradiol. 2006;27(1):13–9. secondary prevention of ischaemic cerebrovascular disease. Clin Investig 25. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Arterioscler. 2020;32(4):175–82. Classification of subtype of acute ischemic stroke. Definitions for use in a 7. Rudd JH, Fayad ZA. Imaging atherosclerotic plaque inflammation. Nat multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treat‑ Clin Pract Cardiovasc Med. 2008;5(Suppl 2):S11–7. ment. Stroke. 1993;24(1):35–41. 8. Kelly PJ, Camps-Renom P, Giannotti N, Martí-Fàbregas J, Murphy S, 26. Norris SL, Grothaus LC, Buchner DM, Pratt M. Effectiveness of physician- McNulty J, et al. Carotid plaque inflammation imaged by 18F-fluorodeox‑ based assessment and counseling for exercise in a staff model HMO. Prev yglucose positron emission tomography and risk of early recurrent stroke. Med. 2000;30:513–23. Stroke. 2019;50(7):1766–73. 27. Martínez-González MA, García-Arellano A, Toledo E, Salas-Salvadó J, Buil- 9. Kelly PJ, Camps-Renom P, Giannotti N, Martí-Fàbregas J, McNulty JP, Cosiales P, Corella D, et al. A 14-item Mediterranean diet assessment tool Baron JC, et al. A risk score including carotid plaque inflammation and and obesity indexes among high-risk subjects: the PREDIMED trial. PLoS stenosis severity improves identification of recurrent stroke. Stroke. ONE. 2012;7(8):e43134. 2020;51(3):838–45. 28. Kernan WN, Ovbiagele B, Black HR, Bravata DM, Chimowitz MI, Ezekowitz 10. Puig N, Camps-Renom P, Camacho M, Aguilera-Simón A, Jiménez-Altayó MD, et al. Guidelines for the prevention of stroke in patients with stroke F, Fernández-León A, et al. Plasma sICAM-1 as a biomarker of carotid and transient ischemic attack: a guideline for healthcare profession‑ plaque inflammation in patients with a recent ischemic stroke. Transl als from the American Heart Association/American Stroke Association. Stroke Res. 2022;13(5):745–56. Stroke. 2014;45(7):2160–236. 11. de Gonzalo-Calvo D, Elosua R, Vea A, Subirana I, Sayols-Baixeras S, Mar‑ 29. US Preventive Services Task Force, Mangione CM, Barry MJ, Nicholson WK, rugat J, et al. Soluble low-density lipoprotein receptor-related protein 1 Cabana M, Chelmow D, Coker TR, et al. Statin use for the primary preven‑ as a biomarker of coronary risk: Predictive capacity and association with tion of cardiovascular disease in adults: US preventive services task force clinical events. Atherosclerosis. 2019;287:93–9. recommendation statement. JAMA. 2022;328(8):746–53. 12. Quinn KA, Pye VJ, Dai YP, Chesterman CN, Owensby DA. Characteriza‑ 30. Luoma J, Hiltunen T, Särkioja T, Moestrup SK, Gliemann J, Kodama T, et al. tion of the soluble form of the low density lipoprotein receptor-related Expression of alpha 2-macroglobulin receptor/low density lipoprotein protein (LRP). Exp Cell Res. 1999;251(2):433–41. receptor-related protein and scavenger receptor in human atheroscle‑ 13. Liu Q, Zhang J, Tran H, Verbeek MM, Reiss K, Estus S, et al. LRP1 shedding rotic lesions. J Clin Invest. 1994;93(5):2014–21. in human brain: roles of ADAM10 and ADAM17. Mol Neurodegener. 31. Llorente-Cortés V, Otero-Viñas M, Hurt-Camejo E, Martínez-González J, 2009;4:17. Badimon L. Human coronary smooth muscle cells internalize versican- 14. Chen J, Pi S, Yu C, Shi H, Liu Y, Guo X, et al. sLRP1 (soluble low-density modified LDL through LDL receptor-related protein and LDL receptors. lipoprotein receptor-related protein 1): a novel biomarker for P2Y12 (P2Y Arterioscler Thromb Vasc Biol. 2002;22(3):387–93. purinoceptor 12) receptor expression in atherosclerotic plaques. Arterio‑ 32. Llorente-Cortés V, Otero-Viñas M, Berrozpe M, Badimon L. Intracellular scler Thromb Vasc Biol. 2020;40(6):e166–79. lipid accumulation, low-density lipoprotein receptor-related protein 15. de Gonzalo-Calvo D, Cenarro A, Martínez-Bujidos M, Badimon L, Bayes- expression, and cell survival in vascular smooth muscle cells derived Genis A, Ordonez-Llanos J, et al. Circulating soluble low-density lipopro‑ from normal and atherosclerotic human coronaries. Eur J Clin Invest. tein receptor-related protein 1 (sLRP1) concentration is associated with 2004;34(3):182–90. hypercholesterolemia: a new potential biomarker for atherosclerosis. Int J 33. de Gonzalo-Calvo D, Colom C, Vilades D, Rivas-Urbina A, Moustafa AH, Cardiol. 2015;201:20–9. Pérez-Cuellar M, et al. Soluble LRP1 is an independent biomarker of 16. Polavarapu R, Gongora MC, Yi H, Ranganthan S, Lawrence DA, Strick‑ epicardial fat volume in patients with type 1 diabetes mellitus. Sci Rep. land D, et al. Tissue-type plasminogen activator-mediated shedding of 2018;8(1):1054. astrocytic low-density lipoprotein receptor-related protein increases the 34. de Gonzalo-Calvo D, Vilades D, Nasarre L, Carreras F, Leta R, Garcia-Moll X, permeability of the neurovascular unit. Blood. 2007;109(8):3270–8. et al. Circulating levels of soluble low-density lipoprotein receptor-related 17. Gorovoy M, Gaultier A, Campana WM, Firestein GS, Gonias SL. Inflamma‑ protein 1 (sLRP1) as novel biomarker of epicardial adipose tissue. Int J tory mediators promote production of shed LRP1/CD91, which regulates Cardiol. 2016;223:371–3. cell signaling and cytokine expression by macrophages. J Leukoc Biol. 35. Lehti S, Nguyen SD, Belevich I, Vihinen H, Heikkilä HM, Soliymani R, et al. 2010;88(4):769–78. Extracellular lipids accumulate in human carotid arteries as distinct three-dimensional structures and have proinflammatory properties. Am J Pathol. 2018;188(2):525–38. Garcia et al. Journal of Translational Medicine (2023) 21:131 Page 10 of 10 36. Allahverdian S, Chehroudi AC, McManus BM, Abraham T, Francis GA. Contribution of intimal smooth muscle cells to cholesterol accumula‑ tion and macrophage-like cells in human atherosclerosis. Circulation. 2014;129(15):1551–9. 37. Allahverdian S, Chaabane C, Boukais K, Francis GA, Bochaton-Piallat ML. Smooth muscle cell fate and plasticity in atherosclerosis. Cardiovasc Res. 2018;114(4):540–50. 38. Iwaki T, Mizuma H, Hokamura K, Onoe H, Umemura K. [18F]FDG uptake in the aortic wall smooth muscle of atherosclerotic plaques in the simian atherosclerosis model. Biomed Res Int. 2016;2016:8609274. 39. Yun M, Yeh D, Araujo LI, Jang S, Newberg A, Alavi A. F-18 FDG uptake in the large arteries: a new observation. Clin Nucl Med. 2001;26(4):314–9. 40. Visser M, Bouter LM, McQuillan GM, Wener MH, Harris TB. Elevated C-reactive protein levels in overweight and obese adults. JAMA. 1999;282(22):2131–5. 41. Nonin S, Iwata S, Sugioka K, Fujita S, Norioka N, Ito A, et al. Plaque surface irregularity and calcification length within carotid plaque predict second‑ ary events in patients with coronary artery disease. Atherosclerosis. 2017;256:29–34. 42. Bytyçi I, Shenouda R, Wester P, Henein MY. Carotid atherosclerosis in pre‑ dicting coronary artery disease: a systematic review and meta-analysis. Arterioscler Thromb Vasc Biol. 2021;41(4):e224–37. 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