antioxidants Article Electronegative LDL Is Associated with Plaque Vulnerability in Patients with Ischemic Stroke and Carotid Atherosclerosis Núria Puig 1,2 , Pol Camps-Renom 3, * , Arnau Solé 1,2 , Ana Aguilera-Simón 2,3 , Elena Jiménez-Xarrié 3 , Alejandro Fernández-León 4 , Mercedes Camacho 5 , Marina Guasch-Jiménez 3 , Rebeca Marin 3 , Joan Martí-Fàbregas 3 , Alejandro Martínez-Domeño 3 , Luis Prats-Sánchez 3 , Francesca Casoni 6 , Belén Pérez 7,8 , Francesc Jiménez-Altayó 7,8 , Jose Luis Sánchez-Quesada 1,9, * and Sonia Benitez 1,9, * 1 Cardiovascular Biochemistry, Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU), 08041 Barcelona, Spain 2 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Stroke Unit, Department of Neurology, Hospital de La Santa Creu i Sant Pau, IIB SANT PAU, 08041 Barcelona, Spain 4 Department of Nuclear Medicine, Hospital de la Santa Creu i Sant Pau, IIB SANT PAU, 08041 Barcelona, Spain 5 Genetic of Complexes Diseases, Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU), 08041 Barcelona, Spain 6 Neurology-Sleep Disorders Center, Department of Clinical Neurosciences, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy 7 Therapeutics and Toxicology, Department of Pharmacology, School of Medicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 8 Neuroscience Institute, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 9 CIBER of Diabetes and Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: pcamps@santpau.cat (P.C.-R.); jsanchezq@santpau.cat (J.L.S.-Q.); sbenitez@santpau.cat (S.B.) Abstract: Owing to the high risk of recurrence, identifying indicators of carotid plaque vulnerability Citation: Puig, N.; Camps-Renom, P.; in atherothrombotic ischemic stroke is essential. In this study, we aimed to identify modified LDLs Solé, A.; Aguilera-Simón, A.; and antioxidant enzymes associated with plaque vulnerability in plasma from patients with a recent Jiménez-Xarrié, E.; Fernández-León, ischemic stroke and carotid atherosclerosis. Patients underwent an ultrasound, a CT-angiography, A.; Camacho, M.; Guasch-Jiménez, and an 18 F-FDG PET. A blood sample was obtained from patients (n = 64, 57.8% with stenosis M.; Marin, R.; Martí-Fàbregas, J.; et al. ≥50%) and healthy controls (n = 24). Compared to the controls, patients showed lower levels of Electronegative LDL Is Associated total cholesterol, LDL cholesterol, HDL cholesterol, apolipoprotein B (apoB), apoA-I, apoA-II, and with Plaque Vulnerability in Patients apoE, and higher levels of apoJ. Patients showed lower platelet-activating factor acetylhydrolase with Ischemic Stroke and Carotid (PAF-AH) and paraoxonase-1 (PON-1) enzymatic activities in HDL, and higher plasma levels of Atherosclerosis. Antioxidants 2023, 12, oxidized LDL (oxLDL) and electronegative LDL (LDL(−)). The only difference between patients 438. https://doi.org/10.3390/ with stenosis ≥50% and <50% was the proportion of LDL(−). In a multivariable logistic regression antiox12020438 analysis, the levels of LDL(−), but not of oxLDL, were independently associated with the degree of Academic Editor: Reto Asmis carotid stenosis (OR: 5.40, CI: 1.15–25.44, p < 0.033), the presence of hypoechoic plaque (OR: 7.52, CI: 1.26–44.83, p < 0.027), and of diffuse neovessels (OR: 10.77, CI: 1.21–95.93, p < 0.033), indicating that Received: 20 December 2022 Revised: 6 February 2023 an increased proportion of LDL(−) is associated with vulnerable atherosclerotic plaque. Accepted: 8 February 2023 Published: 10 February 2023 Keywords: electronegative LDL (LDL(−)); oxidized LDL (oxLDL); ischemic stroke; carotid atherosclerosis; plaque vulnerability Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Approximately 20% of all ischemic strokes are attributed to atherosclerosis, mainly in distributed under the terms and the internal carotid artery. The degree of stenosis is a well-recognized marker of carotid conditions of the Creative Commons plaque instability and of the risk of recurrence, and currently determines the decision Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ whether to perform carotid revascularization in most cases [1]. However, the degree of 4.0/). stenosis alone is not sufficient to make this decision in some frequent clinical situations [2]. Antioxidants 2023, 12, 438. https://doi.org/10.3390/antiox12020438 https://www.mdpi.com/journal/antioxidants Antioxidants 2023, 12, 438 2 of 17 Validated markers for monitoring vascular risk and the response to treatment of these patients are lacking. Therefore, the study of new imaging and plasma biomarkers that complement and improve the assessment of carotid plaque vulnerability in patients with ischemic stroke is essential and may set the basis for future clinical trials. Imaging markers may predict the risk of recurrence beyond the degree of stenosis. Some examples include plaque echoluceny, intraplaque neovascularization, intraplaque hemorrhage, or inflammation assessed by positron emission tomography with 18 F-fluorodeoxyglucose (18 F-FDG PET) [3,4]. However, studies describing plasma biomark- ers related to plaque vulnerability are still scarce. A recent study has revealed the increase of several plasma inflammatory markers in ischemic stroke patients with carotid atheroscle- rosis [5]. Among them, soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), and fractalkine (FKN) were associated with carotid plaque inflammation evaluated by 18 F-FDG PET. sICAM-1 was also associated with stroke recurrence [5]. Another important study demonstrated that baseline levels of IL-6 were associated with plaque vulnerability and progression [6]. Owing to their contribution to inflammation and oxidative stress, the role of lipopro- teins, mainly modified forms of low-density lipoprotein (LDL), in the progression of atherosclerosis is widely established [7]. Several studies demonstrate that increased LDL cholesterol (LDLc) level influences vascular risk, including atherothrombotic stroke [8,9]. In contrast, high-density lipoprotein cholesterol (HDLc) levels are inversely associated with vascular risk and with stroke recurrence [10,11]. However, therapies that increase HDL or decrease LDL are not enough to eliminate vascular risk, a fact that reveals the need to look for biomarkers related to lipoprotein functionality. Beyond LDLc, the presence of modified LDL, such as oxidized LDL (oxLDL), in plasma and atherosclerotic plaque determines the susceptibility to developing vulnerable lesions [12,13]. Several studies have shown an association between circulating oxLDL and atherosclerotic plaque [12,14], and with the presence and prognosis of stroke [15,16]. Electronegative LDL (LDL(−)) is a minor plasma form of modified LDL with inflammatory properties whose proportion is increased in acute myocardial infarction [17], in acute ischemic stroke [18], and in pathologies associated with vascular risk, such as dyslipemias or diabetes [19], and is associated with the extent of carotid stenosis [20]. Unlike LDL, HDL exerts atheroprotective actions, such as the induction of choles- terol efflux (ChE) from tissues [21,22], and anti-oxidant and anti-inflammatory proper- ties [23]. Two enzymatic activities contained in HDL contribute to these properties: platelet- activating factor acetylhydrolase (PAF-AH), also known as lipoprotein-associated phos- pholipase A2, and paraoxonase-1 (PON-1) [24,25]. The functionality of lipoproteins also relies on the content of lipids and apolipoproteins (apo). Therefore, we evaluate in serum from ischemic stroke patients with carotid atherosclerosis: (1) concentrations of lipids and apolipoproteins; (2) anti-oxidant and atheroprotective activities associated with HDL; (3) levels of modified LDL in circulation; and (4) the association of these parameters with features of carotid plaque vulnerability, evaluated by imaging techniques. Altogether, the aim of the present study was to assess which molecules are putative indicators of vulnerability features of the carotid plaque in ischemic stroke patients. 2. Materials and Methods 2.1. Study Design An observational cohort study (NCT03218527) of consecutive adult patients who had had a recent anterior circulation ischemic stroke and carotid atherosclerosis was conducted in the Hospital Santa Creu i Sant Pau between January 2016–March 2019. A control group of healthy subjects was also included. The study was approved by the Ethics Committee of the Hospital (IIBSP-LRB-2017-54, 26 June 2017), and the patients or their legal representatives gave written consent to participate. The study was performed in accordance with the Helsinki Declaration. Antioxidants 2023, 12, 438 3 of 17 2.2. Study Population Patients were included in the study if they fulfilled the following criteria: (1) age ≥ 50 years; (2) anterior circulation ischemic stroke or transient ischemic attack (TIA) within 7 days before inclusion; (3) at least one atherosclerotic plaque in the internal carotid artery (ICA) on the side consistent with stroke symptoms, regardless of the degree of stenosis. Carotid stenosis was graded using the NASCET approach [26] with computed tomography (CT) or magnetic resonance imaging-angiography, as well as being based on hemodynamic criteria using ultrasound [27]; and (4) previous modified Rankin Scale (mRS) score <4. The exclusion criteria were: (1) presence of cardioembolic, lacunar, or unusual stroke etiology according to the TOAST criteria [28]; (2) presence of a hemodynamic stroke/TIA; (3) prior carotid surgery or stenting; (4) presence of comorbidities conditioning a life-expectancy <1 year; (5) concomitant infections at the time of blood extraction; and (6) total artery occlusion. Healthy controls fulfilled the following criteria: (1) age ≥ 50 years, (2) no prior history of ischemic heart disease, and (3) no prior stroke. The following clinical variables were recorded for all of the patients: (1) age and sex; (2) past medical history including hypertension, diabetes, dyslipidemia, prior stroke/TIA, coronary artery disease, tobacco, and alcohol consumption; (3) previous treatments; (4) Na- tional Institutes of Health Stroke Scale (NIHSS) score, as a surrogate of infarct size; (5) body mass index (BMI); (6) regular physical exercise according to the physician-based assess- ment and counseling for exercise (PACE) scale [29]; (7) Mediterranean diet adherence according to the PREDIMED score [30]; (8) mRS score at inclusion; (9) stroke etiology according to the TOAST criteria [28] after a diagnostic work-up that included at least a 24-h-electrocardiogram, an echocardiogram, and an ultrasound carotid examination (assessing plaque echolucency, plaque surface, and degree of stenosis by hemodynamic cri- teria); and (10) results from the admission blood test, including renal function, hemogram, hemostasis, and lipid profile. After the stroke, the treating clinicians provided medical and revascularization treatments according to guidelines [31]. All the healthy controls underwent a clinical interview to assess demographics, lifestyle habits, and prior treatments. Additionally, a standard B-mode and color-Doppler carotid ultrasound examination was performed to rule out the presence of asymptomatic carotid stenosis in both ICA and an electrocardiogram to rule out silent ischemic heart disease and atrial fibrillation. Healthy subjects did not undergo 18 F-FDG PET examination. 2.3. Carotid Plaque Imaging All stroke patients included in the study underwent a carotid ultrasound, a CT- angiography or MR-angiography, and an 18 F-FDG PET/CT within 15 days from the index stroke. The ultrasound protocol included a duplex examination and a Contrast-Enhanced Ultrasound Study (CEUS) to determine the presence of neovascularization. Inflammation of the carotid plaques was assessed by 18 F-FDG PET according to the maximum Standardized Uptake Value (SUV) within the plaque. These imaging protocols are detailed below. 2.3.1. Carotid Ultrasound Protocol All of the patients underwent a carotid ultrasound (US) exam, including a Contrast- Enhanced Ultrasound (CEUS) study, within 15 days from the occurrence of the index stroke. An experienced examiner, P.C.-R., certified in Neurosonology by the Spanish Society of Neurology, performed the US examinations using a Philips CX50® Ultrasound Machine (Philips, Amsterdam, Netherlands) with a linear probe. The US study protocol consisted of two parts: a standard B-mode and color-Doppler carotid plaque characterization, and the CEUS examination. The extracranial common carotid arteries and the ICAs were examined in the longitu- dinal and the transverse planes. A plaque was defined as a localized lumen narrowing of ≥1.5 mm or an increase of >50% in the intima-media thickness compared to the adjacent Antioxidants 2023, 12, 438 4 of 17 portion of the vessel wall. When a plaque was identified, the following sonographic vari- ables were recorded: (1) morphology of the plaque (concentric or eccentric); (2) echogenic- ity of the plaque classified as I = uniformly hypoechoic, II = predominantly hypoechoic, III = predominantly hyperechoic, IV = uniformly hyperechoic; and V = calcified plaque [32]; and (3) degree of stenosis by hemodynamic criteria [27]. Only the largest plaque was stud- ied when patients presented more than one plaque in the ICA. For the statistical analysis, the echogenicity of the plaque was also classified as predominantly hypoechoic (<50% of the surface, comprising I and II categories) and predominantly hyperechoic (≥50% of the surface, comprising III, IV, and V categories). After the B-mode and color-Doppler characterization, the CEUS examination was performed using the preset real-time, contrast-enhanced imaging modality with coded pulse inversion from the Philips CX50® Ultrasound Machine. This setting decreases the mechanical index to 0.1, obtaining an almost completely black screen in the absence of contrast. Then, a bolus of 2 mL of Sonovue® contrast was injected into a peripheral vein and flushed with 10 mL of saline according to the recommendations of the manufacturer (Bracco Imaging, Milan, Italy). At that point, the lumen was filled with the hyperechoic bubbles of the contrast defining the perimeter of the plaque in negative. Time gain compensation was adjusted to achieve homogeneous signal intensity. Finally, a DICOM cine loop was recorded for 120 s starting when the contrast bolus was injected and plaque neovessels were identified as hyperechoic bubbles appearing within the plaque perimeter. The neovascularization of plaque was classified into grades: 0 (no visible microbubbles within the plaque), 1 (moderate microbubbles confined to the shoulder and/or adventitial side of the plaque), and 2 (diffuse microbubbles throughout the plaque), as previously described [33]. This grading was performed by P.C.-R. and by F.C., who was blinded to all of the clinical information, to calculate the interrater agreement and the Cohen’s kappa coefficient. When a discrepancy was detected, the images were reviewed, and a consensus between raters was required. A plaque was suitable for analysis of neovascularization if a DICOM cine loop of 15 s presented enough quality without movement artifact (for example swallowing) and at least 50% of the plaque was visible without calcium shadows. 2.3.2. Carotid 18 F-FDG PET/CT Protocol Carotid 18 F-FDG PET was performed in a Philips Gemini TF TOF 64 PET/CT (Philips Medical System, Eindhoven, The Netherland). The examinations were performed after a fast that lasted a minimum of six hours. PET scans were not performed if pre-PET blood glucose exceeded 10 mmol/L. Two hours before image acquisition, 320 MBq of 18 F-FDG was administered. The uptake phase was standardized with the patient resting. PET images were acquired in a 3-dimensional mode in 2-bed positions for 10 min each. Images from CT angiography and PET were co-registered afterwards to assess the slice of maximal plaque stenosis. 18 F-FDG activity was measured in 10 regions of interest, which were defined relative to the slice of maximal stenosis on the co-registered CT angiography, corresponding to a 1 mm axial plaque slice (5 distal and 5 proximal). 18 F-FDG was quantified using standardized uptake values (SUV g/mL, defined as measured uptake [MBq/mL]/injected dose [MBq] per patient weight [g]). We defined the single hottest slice as the axial slice with maximal SUV uptake (SUVmax) [4]. 2.4. Collection of Blood Samples Peripheral blood samples from the stroke patient were collected on day 7 ± 1 from the stroke. Plasma was collected in ethylenediaminetetraacetic acid (EDTA)-containing Vacutainers and serum in Serum Separator Tubes with clot activator. The tubes were centrifuged at 1500× g for 15 min at 4 ◦ C, and the aliquots were frozen at −80 ◦ C. Antioxidants 2023, 12, 438 5 of 17 2.5. Serum Determinations The lipid profile and apolipoprotein concentrations included total cholesterol, triglyc- erides, non-esterified fatty acids (NEFAs), apoB, apoA-I, apoA-II, apoC-III, apoE, apoJ, very-low-density lipoprotein cholesterol (VLDLc), LDLc, HDLc, and oxLDL. The choles- terol of lipoprotein fractions was quantified by using a direct HDLc method (HDL-C plus, Abbott Core Laboratory, Chicago, IL, USA). Total cholesterol, triglycerides, apoB, and HDLc kits were from Abbott, and were measured in an Alinity ci-series autoanalyzer (Ab- bott). NEFA (Wako Chemicals, Osaka, Japan), apoA-I (Roche, Basel, Switzerland), apoA-II, apoC-III, and apoE (Kamiya Biomedical, Seattle, WA, USA) were measured in a Cobas 6000/c501 autoanalyzer (Roche). ApoJ and oxLDL were quantified by ELISA kits (Mabtech, Stockholm, Sweden and R&D Systems, Minneapolis, MN, USA, respectively) according to manufacturer’s instructions. Parameters related to oxidation, malondialdehyde (MDA) levels and antioxidant capacity, were measured by the MDA/thiobarbituric test and by the 2,2-diphenyl-1- picrylhydrazyl (DPPH) test, respectively. Nitrite levels in serum were measured by the Griess method. PAF-AH activity was quantified using 2-thio-PAF (Cayman Chemicals, Denver, CO, USA) as a substrate [34] and PON-1 activity by using phenylacetate (Sigma/Merck, Darm- stadt, Germany), as described [35]. Total PAF-AH activity was measured in serum. In addition, both PAF-AH and PON-1 activities, as well as ChE, detailed hereafter, were also determined in apoB-depleted serum (HDL fraction) obtained by serum precipitation with dextran sulfate [36]. 2.6. LDL(−) Quantification Total LDL from stroke patients and controls was isolated from plasma by sequential flotation ultracentrifugation (density = 1.019–1.063 g/mL) and dialyzed against buffer A (Tris 10 mM, EDTA 1 mM, pH = 7.4). LDL(−) was isolated from 40 µg of total LDL and its proportion was quantified by using stepwise anion-exchange chromatography in a MonoQ 5/50 GL column (GE Healthcare, Chicago, IL, USA), as described [37]. 2.7. Effect of Lipoproteins on Cells Cell lines THP1-XBlue-MD2-CD14 monocytes (THP1-CD14) (Invivogen, San Diego, CA, USA) and endothelial cells Human Primary Coronary Artery Endothelial Cells (HCAEC) (ATCC, Manassas, VA, USA) were grown following the manufacturer’s recommendations. The growth medium for THP1-CD14 was RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and, 1% Penicillin-Streptomycin from Biowest (Nuaille, France) and with NormocinTM (50 mg/mL), ZeocinTM (100 mg/mL), and G418 (100 mg/mL) antibiotics from Invivogen (San Diego, CA, USA). The growth medium for HCAEC was Vascular Cell Basal Medium, supplemented with the Endothelial Cell Growth Kit VEGF (ATCC, Manassas, VA, USA) which contains 2% of FBS. The experiments were performed as described below. 2.7.1. ChE Capacity of HDL The ChE promoted by 1% of apoB-depleted serum from patients and controls after 24 h’ incubation was determined in THP1-CD14 macrophages using fluorescent-labeled cholesterol, as described [38]. Briefly, THP1-CD14 monocytes were seeded at 200,000 cells/well with RPMI growth medium supplemented with phorbol 12-myristate 13-acetate (PMA) at 50 µg/L for 24 h. Afterwards, cholesterol (0.125 mM) and 20% of fluorescent cholesterol, topFluor-cholesterol linked to boron dipyrromethene (Avanti Polar Lipids, Alabaster, AL, USA), were added for 1 h. Then, cells were pre-treated with the liver X receptor agonist T0901317 (4 µmol/L) (Cayman Chemicals) for 18 h to stimulate efflux pathways. Macrophages were then incubated with 1% of apoB-depleted serum from patients and controls for 24 h. Finally, in order to calculate the efflux capacity, the fluorescence (λEx/Em = 485/530 nm) was Antioxidants 2023, 12, 438 6 of 17 measured by using a Synergy HT spectrophotometer (BioTek, Winooski, VT, USA) in both the supernatant and cells. 2.7.2. Inflammatory Effect of LDL(−) on Cells Total LDL was isolated from pooled plasma from normolipidemic volunteers by ultracentrifugation, and was separated in native LDL (LDL(+)) and LDL(−) by anion- exchange chromatography in an AKTA-FPLC system (GE Healthcare, Chicago, IL, USA). THP1-CD14 monocytes were seeded (400,000 cells/well) with growth medium sup- plemented with PMA at 50 µg/L for 24 h to induce the differentiation into macrophages. HCAEC were seeded (50,000 cells/well) with growth medium for 24 h previously to the experiments. THP1-CD14 macrophages and HCAEC were incubated in the presence or absence of LDL(−) and LDL(+) at 60 mg apoB/L for 24 h with RPMI 1640 medium sup- plemented by 1% FBS and 1% Penicillin-Streptomycin (THP1-CD14) and with Vascular Cell Basal Medium supplemented by the Endothelial Cell Growth Kit VEGF without FBS (HCAEC). After incubation, cell supernatants were collected, and sICAM−1, sVCAM−1, and FKN release were evaluated by ELISA (R&D Systems, Minneapolis, MN, USA). 2.8. Outcomes The primary outcome was finding alterations in the concentrations and functionality of lipoproteins and lipoprotein-related molecules in serum/plasma from ischemic stroke patients versus controls. Secondary outcomes included the association of altered parameters with vulnerability features of the carotid plaque, including degree of stenosis, echolucency, neovascularization, and inflammation. 2.9. Statistical Analysis Continuous descriptive variables were reported as means and standard deviations (SD) or medians (md) and interquartile ranges (IQR) if they were not normally distributed. Categorical variables were expressed as counts and percentages. Differences between groups of patients (more than 2 groups) were assessed using Kruskal–Wallis rank-sum test. Differences on clinical characteristics and lipid parameters were analyzed among the stroke population, divided into two groups < 50% stenosis and ≥50% stenosis, and between all the stroke patients and the control group using the Student’s t-test or the Wilcoxon rank-sum test (when a non-parametric test was required) for continuous variables, and the χ2 test for categorical variables. Continuous variables not-normally distributed as assessed by the Shapiro–Wilk test were log-transformed to approach normality. Correlations between parameters were analyzed using the Spearman’s correlation test. To estimate the association between the proportion of LDL(−) and the degree of plaque stenosis and other plaque features, a multivariable logistic regression analysis was performed using a backward stepwise selection modeling approach. Receiver Operating Characteristic (ROC) curve analyses were performed to compare the predictive value between LDL(−) and oxLDL. Statistical significance for all the analyses was set at p < 0.05 (two-sided). Analyses were performed using Stata v.15 (StataCorp, College Station, TX, USA). 3. Results 3.1. Study Population The study population included 37 patients with at least one atherosclerotic plaque causing ≥50% of stenosis, 27 patients with carotid stenosis <50%, and 27 healthy controls. The clinical characteristics of these groups are detailed in Supplementary Table S1. The groups were balanced for demographics and lifestyle habits except for current smoking and exercise, which were, respectively, more and less frequent in the stroke patients. While the patients’ BMI was lower, the presence of diabetes and treatment with antiplatelet agents Antioxidants 2023, 12, 438 7 of 17 and statins were higher in the stroke patients. In the ≥50% stenosis group, hypertension was less frequent than in the <50% group. 3.2. Lipid Profile and Lipoprotein-Related Molecules Table 1 shows the lipid profile and lipid-related parameters in plasma from stroke patients and from healthy patients. No changes were found according to the degree of stenosis; however, differences existed between all patients and controls. Patients showed decreased levels of total cholesterol, LDLc, and HDLc. Accordingly, they showed a decrease in the main apolipoproteins of LDL and HDL, apoB and apoA-I, respectively, and in the minor apolipoproteins apoA-II and apoE, but no significant differences in apoC-III. Serum from patients showed higher apoJ concentration and lower PAF-AH activity than that from healthy subjects. Table 1. Concentration of lipid profile and lipoprotein-related parameters according to the study population. <50% Group ≥50% Group p All Stroke Control Group p (n = 27) (n = 37) Patients (n = 64) (n = 27) Triglycerides (mM), md (IQR) 1.11 (0.88–1.53) 1.20 (0.94–1.63) 0.601 1.17 (0.92–1.61) 1.18 (0.90–1.45) 0.212 Total cholesterol (mM), 3.80 ± 1.03 3.95 ± 1.186 0.588 3.75 (2.91–4.65) 4.64 (4.00–5.88) <0.001 m ± sd/md (IQR) VLDLc (mM), md (IQR) 0.22 (0.18–0.31) 0.24 (0.19–0.33) 0.601 0.23 (0.18–0.32) 0.24 (0.18–0.29) 0.524 LDLc (mM), md (IQR) 2.48 (1.76–3.48) 2.31 (1.88–3.20) 0.949 2.41 (1.82–3.29) 3.09 (2.58–3.91) <0.001 HDLc (mM), m ± sd/md (IQR) 1.07 ± 0.37 1.02 ± 0.28 0.541 1.03 (0.80–1.32) 1.34 (1.15–1.59) <0.001 LDLc/HDLc ratio, md (IQR) 2.42 (1.85–2.93) 2.57 (1.98–3.11) 0.510 2.48 (1.92–3.03) 2.25 (1.98–2.80) 0.507 NEFA (mM), md (IQR) 0.47 (0.30–0.69) 0.38 (0.26–0.57) 0.404 0.42 (0.27–0.61) 0.43 (0.24–0.48) 0.621 apoB (g/L), md (IQR) 0.68 (0.56–0.90) 0.66 (0.60–0.80) 0.826 0.68 (0.58–0.83) 0.91 (0.78–1.02) <0.001 apoA-I (g/L), md (IQR) 1.22 (1.14–1.50) 1.22 (1.10–1.47) 0.703 1.22 (1.10–1.47) 1.62 (1.44–1.87) <0.001 apoA-II (g/L), m ± sd 0.30 ± 0.07 0.28 ± 0.08 0.468 0.30 ± 0.08 0.37 ± 0.08 <0.001 apoE (g/L), m ± sd 0.04 ± 0.02 0.04 ± 0.02 0.978 0.04 ± 0.02 0.05 ± 0.02 0.006 apoC-III (g/L), md (IQR) 0.05 (0.03–0.09) 0.05 (0.01–0.11) 0.713 0.05 (0.02–0.10) 0.07 (0.03–0.11) 0.144 apoJ (mg/L), md (IQR) 170 (153–219) 180 (147–218) 0.952 179 (148–218) 145 (114–170) <0.001 Total PAF-AH activity 19.10 ± 3.33 19.07 ± 3.58 0.974 19.08 ± 3.41 21.49 ± 2.66 0.003 (µmol/min*mL) m ± sd VLDLc (very low-density lipoprotein cholesterol); LDLc (low-density lipoprotein cholesterol); HDLc (high-density lipoprotein cholesterol); NEFA (non-esterified fatty acid); apo (apolipoprotein); PAF-AH (platelet-activating factor acetylhydrolases). Student’s t-test or Wilcoxon rank-sum test (when a non-parametric test was required) were used to compare groups; p < 0.05 indicates significant differences. No difference was found between groups, either in MDA or antioxidant capacity (Supplementary Table S2). No difference in the nitrite levels in serum of patients compared to Controls was observed (Supplementary Figure S1). However, the achievement of con- clusive results is hindered by the fact that these parameters were not evaluated in all the individuals because of lack of serum sample. 3.3. Anti-Atherogenic Properties of HDL ApoB-depleted serum was used to analyze the protective properties of HDL. PAF-AH and PON-1 enzymatic activities, both related to the anti-oxidant and anti-inflammatory action of HDL, and the ability of apoB-depleted serum fraction to promote ChE from macrophages was evaluated. HDL-associated PAF-AH activity, PON-1 activity, and ChE were lower in stroke patients than in control subjects, although only PAF-AH and PON-1 reached statistical significance (Figure 1). No statistically significant differences between degree of stenosis <50% and ≥50% were found in these anti-atherogenic properties of HDL (Supplementary Figure S2). No correlation was found between levels of HDLc and PAF-AH activity (Spearman’s rho = 0.14; p = 0.219) and ChE (Spearman’s rho = 0.17; p = 0.220), whereas there was a strong correlation between HDLc and PON-1 activity (Spearman’s rho = 0.48; p < 0.001). The apoJ/PON-1 ratio was increased in patients vs. controls (median apoJ/PON-1 activity = 5.24 [IQR 4.30–6.42] vs. 4.02 [IQR 3.11–4.65]; p < 0.001). ti-atherogenic properties of HDL (Supplementary Figure S2). No correlation was found between levels of HDLc and PAF-AH activity (Spearman’s rho = 0.14; p = 0.219) and ChE (Spearman’s rho = 0.17; p = 0.220), whereas there was a strong correlation between HDLc and PON-1 activity (Spearman’s rho = 0.48; p < 0.001). The apoJ/PON-1 ratio was in- creased in patients vs. controls (median apoJ/PON-1 activity = 5.24 [IQR 4.30–6.42] vs. Antioxidants 2023, 12, 438 8 of 17 4.02 [IQR 3.11–4.65]; p < 0.001). Figure 1. Parameters of HDL functionality. PAF-AH and PON-1 activities and ChE were quantified in Figure 1. Parameters of HDL functionality. PAF-AH and PON-1 activities and ChE were quantified apoB-depleted serum. PAF-AH and PON-1 activities were calculated from the slope of the enzymatic in apoB-depleted serum. PAF-AH and PON-1 activities were calculated from the slope of the en- activity measured by colorimetric detection. (a) PAF-AH activity was determined by using 2-thio-PAF zymatic activity measured by colorimetric detection. (a) PAF-AH activity was determined by using as a substrate. 2-thio-PAF (b) PON-1 as a substrate. (b)activity PON-1was measured activity using phenylacetate was measured as a substrate. using phenylacetate (c) ChE capacity as a substrate. (c) of apoB-depleted serum was analyzed in THP1-CD14 macrophages after 24 h ChE capacity of apoB-depleted serum was analyzed in THP1-CD14 macrophages after 24 h incu-incubation. Patients (n = Patients bation. 46); Controls (n =Controls (n = 46); 26); # vs.(nControls; p 50% stenosis matography. and <50% stenosis. (a) Levels (b) Proportion of oxLDL of LDL(−) when stratifying bythe patients degree werestenosis of carotid divided(w/o intostenosis groupsnof >50% stenosis and50– = 27, <50% n = 27, <50% 69%stenosis. (b) Proportion n = 13, 70–90% of LDL( n = 12, >90% −) Kruskal–Wallis n = 10), stratifying by degree of performed test was carotid stenosis (p (w/o stenosis = 0.0017), # vs. ncontrols = 27, <50% (w/onstenosis); = 27, 50–*69% n = 13, vs. <50% 70–90%p n< = patients; 12, >90% n = 10), Kruskal–Wallis test was performed 0.05. (p = 0.0017), # vs. controls (w/o stenosis); * vs. <50% patients; p < 0.05. 3.5. Association of Plaque Characteristics with Modified LDL 3.5. Association of Plaque Characteristics with Modified LDL Besides stenosis degree, we aimed to study the association between the proportion of LDL(−) and other Besides stenosis features degree, of plaque we aimedincluding vulnerability, to study echolucency, the association between the proportion intraplaque of LDL( − ) and other features of plaque vulnerability, including neovascularization, and inflammation assessed by F-FDG PET/CT. Table 2 shows the 18 echolucency, intraplaque neovascularization, and inflammation assessed by 18 F-FDG PET/CT. Table 2 shows the levels of LDL(−), oxLDL, and LDLc by those characteristics. levels of LDL(−), oxLDL, and LDLc by those characteristics. Table 2. LDL(−), oxLDL, and LDLc levels by characteristics of plaque vulnerability. Echolucency Predominantly Predominantly p Hypoechoic (n = 19) Hyperechoic (n = 45) LDL(−) (%), md (IQR) 9.5 (7.1–11.7) 7.4 (5.6–9.9) 0.010 oxLDL (U/mmol LDLc), md (IQR) 15.7 (5.4–21.6) 11.4 (8.2–17.1) 0.734 LDLc (mM), md (IQR) 3.02 (1.85–3.32) 2.33 (1.76–2.90) 0.228 Intraplaque neovascularization Present (n = 28) Absent (n = 9) p LDL(−) (%), md (IQR) 7.9 (6.5–10.5) 6.5 (3.8–7.6) 0.047 oxLDL (U/mmol LDLc), md (IQR) 13.2 (7.0–17.1) 12.3 (11.6–13.1) 0.976 LDLc (mM), md (IQR) 2.35 (1.89–3.20) 2.71 (2.01–3.70) 0.481 Diffuse intraplaque neovascularization Present (n = 14) Absent (n = 23) p LDL(−) (%), md (IQR) 9.0 (7.1–11.7) 7.0 (4.6–9.2) 0.033 oxLDL (U/mmol LDLc), md (IQR) 15.5 (6.2–17.1) 11.9 (8.8–14.5) 0.569 LDLc (mM), md (IQR) 3.03 (1.90–3.44) 2.40 (1.82–3.1) 0.468 Intraplaque inflammation SUVmax ≥ 2.85 g/mL SUVmax < 2.85 g/mL p (n = 26) (n = 38) LDL(−) (%), md (IQR) 7.2 (5.4–10.9) 8.0 (6.2–10.2) 0.642 oxLDL (U/mmol LDLc), md (IQR) 15.9 (10.1–18.7) 9.9 (7.0–16.9) 0.127 LDLc (mM), md (IQR) 2.26 (1.89–3.18) 2.52 (1.75–3.37) 0.603 oxLDL (oxidized low-density lipoprotein); LDL(−) (electronegative LDL); LDLc (low-density lipoprotein choles- terol); SUVmax (maximal standardized uptake value). The Wilcoxon rank-sum test was used to compare groups; p < 0.05 indicates significant differences. The proportion of LDL(−) was significantly higher in those patients presenting pre- dominantly hypoechoic plaques and intraplaque neovascularization, whilst LDLc and oxLDL did not show association with any of the plaque features analyzed. Multivariable logistic regression analysis was performed to assess the association of LDL(−) with the characteristics of plaque vulnerability. Table 3 shows that LDL(−) was Antioxidants 2023, 12, 438 10 of 17 independently associated with the probability of presenting carotid stenosis ≥50% (a), predominantly hypoechoic plaque (b), and diffuse intraplaque neovascularization (c). Sup- plementary Tables S1, S4 and S5 detail the bivariate analyses of predictors of carotid stenosis ≥50%, predominantly hypoechoic plaque and diffuse intraplaque neovascularization, re- spectively. Table 3. Multivariable logistic regression analysis of predictors of carotid vulnerability. OR 95% CI p (a) Plaque stenosis ≥50% Proportion of LDL(−) 5.40 1.15–25.44 0.033 (×1 increase in the logarithm) Hypertension 0.12 0.01–1.02 0.052 (b) Predominantly hypoechoic plaque Proportion of LDL(−) 7.52 1.26–44.83 0.027 (×1 increase in the logarithm) Prior statin therapy 0.23 0.06–0.79 0.020 (c) Diffuse intraplaque neovascularization Proportion of LDL(−) 10.77 1.21–95.93 0.033 (×1 increase in the logarithm) Antioxidants 2023, 12, x FOR PEER REVIEW 11 of 18 3.6. Prediction of Carotid Plaque Vulnerability According to the Plasma Proportion of LDL(−) We performed ROC analyses to identify a balanced cut-off point of LDL(−) for pre- We performed ROC analyses to identify a balanced cut-off point of LDL(−) for pre- dicting carotid plaque vulnerability with high sensitivity. We observed that a cut-off point dicting carotid plaque vulnerability with high sensitivity. We observed that a cut-off of 6.9% of LDL(−) predicted the risk of presenting carotid stenosis ≥50% with a sensitivity point of 6.9% of LDL(−) predicted the risk of presenting carotid stenosis ≥50% with a of sensitivity 82.9% andofa 82.9% specificity and aof 51.9%, predominantly specificity hypoechoic of 51.9%, predominantly plaque with hypoechoic plaque a sensitivity with a of 94.4% and a sensitivity of 43.2%, and diffuse intraplaque neovascularization sensitivity of 94.4% and a sensitivity of 43.2%, and diffuse intraplaque neovascularization with a sensi- tivity withofa 85.7% andofa 85.7% sensitivity sensitivity and a of 43.5%. Figure sensitivity 4 shows of 43.5%. Figure that LDL( 4 shows −),LDL(−), that but notbutoxLDL, not was a good oxLDL, predictor of such was a good plaque predictor features. of such plaque Byfeatures. using this cut-off By using point this in point cut-off the multivariable in the logistic regression multivariable analyses logistic (adjusted regression analysesby(adjusted the same by variables as in Table the same variables as in3), the 3), Table presence ≥6.9% of the presence of LDL( −) was of ≥6.9% of LDL(−) was independently independently associated associated with thewith the presence presence of ca- of carotid stenosis rotid (OR ≥50% stenosis ≥50% = 4.98, 95%(OR CI= 1.46–16.97, 4.98, 95% CIp 1.46–16.97, = 0.010) and p = of 0.010) and of predominantly predominantly hypoechoic hy-plaque (ORpoechoic = 18.8,plaque 95% CI (OR = 18.8, 95%p CI 2.10–68.40; 2.10–68.40; = 0.009), andp was = 0.009), and significantly almost was almost significantly associated with intraplaque neovascularization (OR = 4.62, 95% CI 0.84–25.49; p0.84–25.49; associated with intraplaque neovascularization (OR = 4.62, 95% CI = 0.079). p = 0.079). (a) (b) Figure Figure4.4.ROC ROCcomparison analysesbetween comparison analyses between LDL(−) −) and LDL(and oxLDL. oxLDL. (a) Prediction (a) Prediction of a carotid of a carotid degree degree of of stenosisgreater stenosis greaterthan than 50%. 50%. (b) (b)Prediction Predictionof of predominantly hypoechoic predominantly plaque. hypoechoic plaque. 3.7. LDL( 3.7. −) as an LDL(−) an Inductor InductorofofInflammation Inflammation in Cultured in Cultured CellsCells Based Basedon onour ourprevious previousfindings findings of of the the association of sICAM-1 association of sICAM-1sVCAM-1 sVCAM-1and andFKN FKN with with plaque plaque inflammation, inflammation, we assessed we assessed whether LDL(− whether LDL(−) induced ) induced the release the release of these of these molecules molecules in cultured macrophages and endothelial cells. Figure 5 shows that LDL(−) did promote that induction, except for FKN in macrophages, which was undetectable. Antioxidants 2023, 12, 438 11 of 17 Antioxidants 2023, 12, x FOR PEER REVIEW 12 of 18 in cultured macrophages and endothelial cells. Figure 5 shows that LDL(−) did promote that induction, except for FKN in macrophages, which was undetectable. Figure5.5.LDL( Figure −)-induced release LDL(−)-induced releaseofofinflammatory inflammatorymolecules moleculesininmacrophages macrophagesand andendothelial endothelialcells. cells. THP1-CD14 THP1-CD14macrophages macrophages(400,000 (400,000cells/well) and HCAEC cells/well) and HCAEC(50,000 (50,000cells/well) cells/well) were were incubated incubated in in the the presence presence or or absence absence of of LDLs LDLs (60(60 mgmg apoB/L) apoB/L) for for 24sICAM-1, 24 h. h. sICAM-1, sVCAM-1, sVCAM-1, and and FKNFKN release release were thenthen were evaluated by ELISA evaluated in theinsupernatant by ELISA of (a,b) the supernatant of THP1-CD14 macrophages (a,b) THP1-CD14 (n = 11) macrophages (nand ofand = 11) (c–e) ofHCAEC (n = 7).(nThe (c–e) HCAEC = 7).Kruskal–Wallis The Kruskal–Wallistest was testperformed was performed(p < 0.01 (p < in allincases), 0.01 # vs.# Blank; all cases), * vs. vs. Blank; LDL(+); p < 0.05. * vs. LDL(+); p < 0.05. 4.4.Discussion Discussion InInthethepresent present study, patients study, patientswith a recent with ischemic a recent ischemicstroke and and stroke carotid atherosclerosis carotid atheroscle- showed increased rosis showed blood levels increased blood of oxLDL levels and LDL( of oxLDL and−LDL(−). ). The proportion of LDL( The proportion of− ), but not LDL(−), but that notofthatoxLDL, was associated of oxLDL, with the was associated withdegree of carotid the degree stenosis, of carotid as wellas stenosis, aswell withas other with features of plaque other features of vulnerability, such as echolucency plaque vulnerability, and neovascularization. such as echolucency In addition,In and neovascularization. we demonstrated that LDL( − ) induced the release of sICAM-1, addition, we demonstrated that LDL(−) induced the release of sICAM-1, sVCAM-1, sVCAM-1, and FKN andin endothelial cells and macrophages. FKN in endothelial cells and macrophages. ItItisiswidely widelyaccepted acceptedthat thatthe themain maininductor inductorofofinflammation inflammationand andlipid lipidaccumulation accumulation ininatherosclerotic atheroscleroticlesionslesionsisismodified modifiedLDL LDLretained retainedininthe theintima intima[7]. [7].Therein, Therein,one oneofofthe the main sources of LDL modification is the oxidative milieu yielded by main sources of LDL modification is the oxidative milieu yielded by radical oxygen spe- radical oxygen species (ROS) and oxidative cies (ROS) enzymes and oxidative released enzymes by cellsby released from the cells arterial from wall. In wall. the arterial this study, In thisoxLDL study, and LDL( − ) were higher in the blood from patients with oxLDL and LDL(−) were higher in the blood from patients with a recent ischemic a recent ischemic strokestroke and carotid atherosclerosis than in healthy controls. Contrariwise, the patients and carotid atherosclerosis than in healthy controls. Contrariwise, the patients showed a showed a more favorable lipid profile more favorable than control lipid profile subjects, than control havinghaving subjects, lower lower levels levels of totalofcholesterol and total cholesterol LDLc. This may be attributable to the known decrease in lipid levels and LDLc. This may be attributable to the known decrease in lipid levels after ischemia after ischemia and to the administration of statin immediately after the ischemic stroke. In line with the and to the administration of statin immediately after the ischemic stroke. In line with the decrease in LDL and HDL, the main apolipoproteins in them (apoB, apoA-I, and apoA-II, decrease in LDL and HDL, the main apolipoproteins in them (apoB, apoA-I, and apoA-II, respectively) and minor apoprotein apoE were also lower in patients. Otherwise, the respectively) and minor apoprotein apoE were also lower in patients. Otherwise, the levels of apoJ were higher, as was the apoJ/PON−1 ratio, and both correlated with oxLDL levels of apoJ were higher, as was the apoJ/PON−1 ratio, and both correlated with oxLDL levels. Accordingly, the apoJ/PON−1 ratio was shown to be elevated in patients with levels. Accordingly, the apoJ/PON−1 ratio was shown to be elevated in patients with cardiovascular risk and in atherosclerotic animal models, as well as in the presence of cardiovascular risk and in atherosclerotic animal models, as well as in the presence of oxLDL [39]. Increased apoJ levels in the plasma of ischemic patients could be interpreted oxLDL [39]. Increased apoJ levels in the plasma of ischemic patients could be interpreted as a response to ischemia, since apoJ is known to act as an acute phase protein [40]. as a response to ischemia, since apoJ is known to act as an acute phase protein [40]. The protective action of HDL was altered in patients, as is suggested by its lower PAF-AH and PON-1 enzymatic activities, and by the trend to lower ChE capacity. This Antioxidants 2023, 12, 438 12 of 17 The protective action of HDL was altered in patients, as is suggested by its lower PAF-AH and PON-1 enzymatic activities, and by the trend to lower ChE capacity. This diminution may be due not only to the lower amount of HDL particles but also to a lack of HDL functionality in patients. The decrease in PON-1 and PAF-AH activities is closely involved in the anti-oxidant and anti-inflammatory properties ascribed to HDL [25], which may contribute to the inflammatory state of patients favoring LDL oxidation. In this regard, the activity of PAF-AH in serum and of PAF-AH associated with HDL correlated with oxLDL levels. Although higher concentrations of oxLDL in serum were found in the stroke patients of our study, no difference was observed in its levels depending on the degree of stenosis or other features of plaque vulnerability. The most significant findings of the current study are related to the predictive value of plasma LDL(−) for plaque vulnerability. To the best of our knowledge, only one previous study had reported increased levels of LDL(−) in ischemic stroke patients [18], despite showing higher proportions than in our study, presumably as a result of methodological differences, mainly the type of patients, the time of blood extraction, and the chromato- graphic method of LDL(−) isolation. Our main highlights are that: (1) Stroke patients had a higher proportion of LDL(−), independently of other lipid-related molecules and of treatment with high-dose statins [41–43]; and (2) LDL(−), but not oxLDL, was inde- pendently associated with the degree of carotid stenosis, hypoechogenicity, and diffuse intraplaque neovascularization, all of which are features of plaque vulnerability. The ability of LDL(−) to induce inflammation and foam cell formation [19,44], as well as the expression of molecules related to angiogenesis in endothelial cells and macrophages [44,45], may underlie these characteristics of carotid plaque. Moreover, LDL(−) shows a high affinity to proteoglycans of the artery wall [46] and hence it is prone to be retained in the subendothe- lial space, where it is able to interact with cell types present in the atherosclerotic lesion. In addition, LDL(−) promotes the release of MMP [47], a fact that is likely contributing to enhanced plaque vulnerability. In our study there was no correlation between LDL(−) and oxLDL levels, and there was neither higher MDA concentration nor antioxidant capacity in serum from patients. In agreement, our previous observations showed no evidence of oxidative modification in LDL(−) compared to native LDL, since they have a similar content of MDA, fatty acid hydroxides, and antioxidants [48]. Previous literature has reported that LDL(−) elicits effects related to oxidative stress in endothelial cells, such as mitochondrial free-radical production and senescence [49], superoxide dismutase expression [50], and inhibition of endothelial nitric oxide synthase activation [51,52]. Notably, in a very recent study conducted in the same cohort of patients, we found that serum from ischemic patients with a high degree (≥70%) of stenosis promoted cyclooxygenase-dependent endothelial dysfunction in carotid arteries of non-ischemic mice [53]. Therefore, the relationship among LDL(−), oxidative stress, and endothelial dysfunction in atherothrombotic stroke patients deserves to be elucidated and is being evaluated in our ongoing investigations. In another recent study, we described how in the same cohort of patients there was an association between carotid plaque inflammation and sICAM-1, sVCAM-1, and FKN concentrations in plasma. Interestingly, the in vitro experiments of the present study showed that LDL(−) promote the release of those molecules in endothelial cells and in macrophages, likely contributing to the inflammatory state within the plaque and to the presence of neovessels, since such molecules are related to angiogenesis [54–56]. We hypothesize, as illustrated in Figure 6, that LDL(−) may be more a cause than a consequence of plaque progression, and hence it mainly acts earlier by promoting plaque vulnerability, but without showing a direct association with the plasma inflammatory molecules released at the onset of stroke and associated with SUVmax [5]. This does not eliminate the possibility that a part of plasma LDL(−) may also be generated by lipolysis/proteolysis in the arterial wall and then released from vulnerable plaque. Antioxidants 2023, 12, 438 13 of 17 Antioxidants 2023, 12, x FOR PEER REVIEW 14 of 18 Figure Figure 6. Role 6. Role ofofLDL( LDL(−) −) inin ischemic ischemicstroke strokeassociated with associated atherosclerosis. with Before atherosclerosis. the stroke Before event, event, the stroke subjects with a high susceptibility to developing carotid atherosclerosis show a high percentage of subjects with a high susceptibility to developing carotid atherosclerosis show a high percentage of LDL(−) with inflammatory properties. At first, LDL(−) contributes to carotid plaque formation and LDL( −) withthe promotes inflammatory progress of the properties. At sustained lesion by the −) contributes first, LDL(induction to carotidand of inflammation plaque formation and lipid accumu- promotes the progress lation. Then, of the as a result lesion of the by the sustained progression inductionthe of atherosclerosis, ofplaque inflammation rupturesand andlipid accumulation. releases part of its Then, as inflammatory a result of the content and modified progression LDLs into the circulation, of atherosclerosis, the plaqueeventually ruptures leading to the part and releases for- of its mation of thrombus and a stroke event. inflammatory content and modified LDLs into the circulation, eventually leading to the formation of thrombus and a stroke event. The present study has some limitations. As the number of patients was small, eval- uation in larger cohorts is required to confirm the findings. In addition, some unresolved The present study has some limitations. As the number of patients was small, evalu- questions emerge from the present results, for example regarding the evaluation of pa- ation in larger cohorts is required to confirm the findings. In addition, some unresolved rameters related to lipid oxidation and antioxidant capacity in plasma. In order to answer questions emerge ongoing those questions, from the present studies results, are being for example conducted in the regarding same cohortthe evaluation of patients. This of pa- rameters relatedistomainly investigation lipid oxidation focused onand antioxidant determining the capacity oxidative in plasma. In parameters in order plasma, toas answer those wellquestions, ongoing as the molecular studies are composition of being conducted the isolated in the and lipoproteins, same thecohort of patients. oxidative proper- This investigation ties of LDL is andmainly focused on protection the anti-oxidative determining the oxidative of HDL against LDL parameters oxidation.in plasma, as well as the molecular composition of the isolated lipoproteins, and the oxidative properties of LDL 5. and Conclusions the anti-oxidative protection of HDL against LDL oxidation. In conclusion, ischemic stroke patients with carotid atherosclerosis, in spite of 5. Conclusions treatment with statin and a favorable lipid profile, showed high levels of LDL(−). This In conclusion, ischemic stroke patients with carotid atherosclerosis, in spite of treat- ment with statin and a favorable lipid profile, showed high levels of LDL(−). This observa- tion, in conjunction with the association with the degree of stenosis, plaque neovascular- ization, and hypoechogenicity, suggests that LDL(−) is a much better indicator of plaque vulnerability than oxLDL. In addition to the increased presence of inflammatory LDL(−), Antioxidants 2023, 12, 438 14 of 17 the lower levels of HDL with less protective enzymes may converge in an enhanced inflam- matory response in these patients. Although further investigation is required to assess its predictive usefulness, our findings suggest that LDL(−) may be useful in the future for the development of therapeutic strategies, in symptomatic and also in asymptomatic patients with carotid atherosclerosis. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12020438/s1. Table S1: Clinical characteristics of the patients; Table S2: Serum MDA levels and antioxidant capacity; Table S3: Correlation between the proportion of LDL(−) and oxLDL with other lipid-related parameters; Table S4: Clinical variables and lipid parameters in patients with predominantly hypoechoic or hyperechoic plaque; Table S5: Clinical variables and lipid parameters in patients with diffuse or non-diffuse intraplaque neovascularization. Figure S1: Nitrite levels in serum; Figure S2: Parameters of HDL functionality. Author Contributions: Conceptualization, N.P., P.C.-R., J.L.S.-Q. and S.B.; methodology, N.P., A.A.-S., M.C., R.M., B.P., F.J.-A. and A.S.; writing—original draft preparation, N.P., P.C.-R., A.S. and S.B.; writing—review and editing, P.C.-R., J.L.S.-Q., J.M.-F., E.J.-X., F.J.-A. and S.B.; acquisition data: A.F.-L., A.M.-D., M.G.-J. and L.P.-S.; analysis or interpretation of data: F.C., N.P., P.C.-R. and S.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants 201716.10 from Fundació La Marató TV3, PID 2020-113634RB-C22 from Ministerio de Ciencia e Innovación, and FIS PI19/00421 and PI20/00334 from the Instituto de Salud Carlos III (co-financed by the European Regional Development Fund). N.P. is funded by the Instituto de Salud Carlos III predoctoral contract FI20/00252. S.B. and JLS- Q. are members of CIBER of Diabetes and Metabolic Diseases (CIBERDEM, CB07/08/0016, Insti- tuto de Salud Carlos III Project). S.B., P.C.-R., J.M.-F. and A.A.-S. are members of RICORS-ICTUS (RD21/0006/0006). S.B., J.L.S.-Q., and N.P. are members of the Quality Research Group 2017-SGR– 1149 from Generalitat de Catalunya, and of the Spanish Atherosclerosis Society Vascular Biology Group. Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU) is accredited by CERCA Pro- gramme/Generalitat de Catalunya. Institutional Review Board Statement: The study was approved by the Ethics Committee of the Hospital de la Santa Creu i Sant Pau (IIBSP-LRB−2017–54, 26 June 2017), and the patients or their legal representatives gave written consent to participate. The study was performed in accordance with the Helsinki Declaration. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data are contained within the article or as supplementary material. Conflicts of Interest: The authors declare no conflict of interest. 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