Complement Factor H and Pro-Resolving Mediators Synergistically Enhance Plaque Stability in Peripheral Arterial Disease

Complement Factor H and Pro-Resolving Mediators Synergistically Enhance Plaque Stability in Peripheral Arterial Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Complement Factor H and Pro-Resolving Mediators Synergistically Enhance Plaque Stability in Peripheral Arterial Disease Eija Nissilä, Shahan Syed, Pavel Uvarov, A. Inkeri Lokki, Mirjami Laivuori, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6828685/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Inflammation is a key driver of plaque rupture and adverse cardiovascular events in atherosclerosis patients. However, accurate identification of high-risk individuals in preventative cardiology has remained elusive. Methods In this study we analyzed the biochemical characteristics of high-density lipoproteins (HDL), plasma metabololipidomics and atherosclerotic plaques obtained from peripheral artery disease patients. This included the measurements of circulating complement activation markers, complement factor H-associated HDL, malondialdehyde modified HDL, plasma lipid mediators, as well as analyzing the HDL and plaque proteome. In addition, arterial plaques were subjected to histological and immunofluorescence staining. Results We identified novel pro- and anti-inflammatory molecules and structural components of arterial plaques indicative of plaque stability. An increase in HDL-associated complement regulatory protein factor H correlated with higher levels of specialized pro-resolving lipid mediators, changes in plaque calcification, and specific extracellular matrix proteins. The presence of factor H in HDL was associated with increased cholesterol efflux supporting its role in HDL’s antiatherogenic effects. Conclusions Our findings indicate that complement regulation and pro-resolving responses work synergistically to exert anti-inflammatory effects, which are essential for maintaining extracellular matrix integrity. These findings may assist in early detection of high-risk cardiovascular disease and optimize therapeutic strategies to prevent adverse events. Complement inflammation lipidomics proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Atherosclerosis, the leading cause of cardiovascular disease, is characterized by the thickening of the arterial wall, which leads to constricted blood flow. It typically co-exists in several arterial beds, and (PAD) increases the risk of coronary artery disease. The immune response and inflammation play crucial roles in the development and progression of atherosclerosis. It can become life-threatening by causing ischemia in affected organs due to restricted blood flow or by triggering plaque rupture, which releases atherosclerotic particles into the bloodstream and raises the risk of a critical thrombus. In either case, early detection is crucial for timely and effective treatment. High-density lipoprotein (HDL) removes cholesterol from the periphery to the liver and promotes macrophage cholesterol efflux. It also has many beneficial anti-inflammatory and anti-oxidative functions that can mitigate the risk factors of cardiovascular diseases. Low levels of HDL-cholesterol (HDL-C) are recognized as a risk factor for PAD [1]. However, cardiovascular health is influenced not only by the quantity of HDL but also by its quality and functionality, which are determined by HDL size, structure, and protein and lipid composition. In addition, various functional capacities of HDL, such as antioxidant, anti-inflammatory, and cholesterol efflux-promoting activities, play a crucial role in determining its impact on vascular health. These properties can be impaired by inflammatory molecules released during metabolic disorders, atherosclerosis, and acute infections [2]. Importantly, the reduced capacity of HDL to promote cholesterol efflux from donor cells correlates with atherosclerotic burden in the coronary and carotid arteries [3]. Apolipoprotein A1 is the major determinant of HDL functionality and its formulation has been evaluated in clinical trials for the reduction of myocardial infarction, stroke, or death from cardiovascular causes. These studies, however, have not led to clinical success [4]. Malondialdehyde-modified high-density lipoproteins (MDA-HDLs) are formed during lipid peroxidation, and they are known to promote inflammatory responses that play a major role in the development of atherosclerosis. Even though the role of MDA-HDL in PAD has thus far not been explored, MDA-modified low-density lipoprotein (MDA-LDL) has been identified as a marker for plaque vulnerability in peripheral artery disease patients [5]. Complement activation is a key driver of inflammation and atherogenesis [6]. This process is regulated by the main fluid phase complement regulator, complement factor H (CFH), which binds to C3b molecules deposited on self-structures and to MDA-modified epitopes in atherosclerotic lesions [7]. On HDL, CFH interacts with apolipoprotein E (ApoE) to confer protection against the complement attack [8]. ApoE4 isoform is strongly associated with increased risk for Alzheimer’s disease and to a lesser extent with coronary heart disease [9]. This isoform has also been shown to confer reduced binding to CFH leading to attenuated complement regulation and increased inflammation [10]. In addition to complement activation, pro-inflammatory lipid mediators are known to promote inflammation in atherosclerotic lesions, while (SPMs) attenuate and help resolve inflammation. Fatty acid-derived SPMs not only counter-regulate inflammation but also increase efferocytosis, reduce aortic plaque size and necrosis, and increase the thickness of the fibrous cap of plaques [11, 12]. Pro-inflammatory conditions often lead to increased lipid mediator production, as evidenced by elevated circulating pro-inflammatory mediator and SPM levels observed, for example, immediately after myocardial infarction. However, imbalance among different types of lipid mediators may impair plaque stability, with low SPMs increasing the risk of cardiovascular events [13]. In the current study, we explored pro- and anti-inflammatory molecules in a cohort of PAD patients. Our biochemical data, including HDL oxidation and complement regulation, along with proteomics data, identified novel lipid-specific markers of HDL and plasma metabololipidome that contribute to anti-inflammatory and anti-atherogenic properties of HDL. Our histological and proteomics data showed that increased levels of these markers were associated with stability markers in femoral artery plaques and a lower artery stenosis grade. Furthermore, the histomorphological features of femoral artery plaques and observations from magnetic resonance angiography correlated with our biochemical findings. Our data suggest that complement regulation and specialized pro-resolving mediators enhance plaque stability by promoting HDL function and pro-resolving responses. These findings provide a potential target for drug development and improved disease diagnostics. METHODS Patient samples and data This study includes a subpopulation of atherosclerosis patient cohort that has been described earlier (Table S1 ) [14, 15]. Femoral plaque samples (n = 6) were collected during endarterectomy of the femoral artery bifurcation and stored at -80°C. Magnetic resonance angiography (MRA) was used to visualize arterial flow patterns, complemented by histological examination of atherosclerotic plaques using H&E-stained paraffin sections to reveal the distinct patterns of the arterial disease (Fig. S1 ). The patients provided written informed consent for participation in the study prior to undergoing endarterectomy surgery. The study protocol was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa (ASO-project 78/13/03/00/2014), and all procedures were conducted in accordance with applicable guidelines and regulations. ApoE genotyping Four microliters of patient DNA (20 ng/µl) extracted from peripheral blood mononuclear cells, were used for sequencing. Previously published primers were used [16] along with with OneTaq Hot Start 2X Master Mix with Standard Buffer (New England Biolabs, Ipswich, MA, USA). The PCR reaction was performed at 98° C for 4 min, followed by 35 cycles of 98° C for 10 sec, 60° C for 30 sec, and 72° C for 40 sec and 72° C for 10 min. PCR products were purified from agarose gels using ExoSAP-IT™ (Thermo Fisher) by incubating at 37° C for 30 min followed by 80° C for 15 min. The products were sequenced twice using forward and reverse primers, electrophoresis was performed using ABI3730xl DNA Analyzer, and base calling was conducted with Sequencing Analysis 7 at Finnish institute for Molecular Medicine (FIMM, University of Helsinki). The sequences were analyzed using Sequencher 4.8 software. Measuring malondialdehyde (MDA) adducts on HDL. HDL samples were isolated from the EDTA plasma obtained from the patients and healthy volunteers [17]. MDA-HDL was measured using Oxidized HDL Assay Kit (Cat.no ab242308, Abcam) from 1:50 diluted precipitated serum samples (n = 17). Absorbances were measured at 450 nm using Hidex Sense microplate reader (Hidex). ELISA HDL (n = 17) was coated onto 96-well microplates at a concentration of 5–20 µg/ml in PBS (Nunc PolySorp, Cat. No. 444865) and incubated overnight at 4 °C. Plates were washed with PBS and blocked with 3% fatty acid-free bovine serum albumin (BSA; Cat.no. P6156, Biowest) in PBS. After washing wells were incubated with goat anti-factor H (1:2000, Cat. No. 341276, Calbiochem), rabbit anti-apoE (1:10000, a kind gift from Dr. Matti Jauhiainen), or rabbit anti-C3c (1:500, Cat. No. OSAP14/15, Behring) antibodies in 0.3% BSA-PBS for 1 hour at + 37 °C. After washing the wells were incubated for 40 minutes at 37 °C with HRP-conjugated anti-goat (1:2000, Cat. No. 705-035-147, Jackson ImmunoResearch laboratories) or anti-rabbit IgG (Cat. No. NEF812, Perkin Elmer) in 0.3% BSA/PBS. After three washes, the wells were incubated with 1-Step™ Ultra TMB-ELISA Substrate Solution (Cat. No. 34028, Thermo Fisher Scientific). The reaction was stopped with 0.5 M H 2 SO 4 , and absorbance was measured at 450 nm. All experiments were conducted in triplicate or duplicate. Perlecan levels were analyzed from 1:10 or 1:20 diluted EDTA (n = 17) or serum samples (n = 35) using a human HSPG (perlecan) ELISA kit (Cat.no. ab274393, Abcam) according to manufacturer’s instructions. Western blot and cholesterol efflux assay The presence of CFH, ApoE, ApoAI or C3b on HDL was determined by Western blotting (Fig. S2). HDL-2 particles (2.8 mg/ml) were incubated with complement proteins and subjected to size-exclusion chromatography and the protein concentrations were measured prior the cholesterol efflux assay. The cholesterol efflux assay was performed according to manufacturer’s instructions (Cat. No. ab196985, Abcam) using THP-1 cells activated with 100nM PMA for 72 h. The cells were incubated with 5–10 µg of HDL. Replicate assays were normalized by averaging the values of all samples within each assay replicate and then multiplying by the calculated ratios. Each experiment was performed in at least duplicate and repeated three times. Immunofluorescence staining of peripheral artery plaques Cryosections (Fig. S3) of femoral plaques were stained using a standard H&E protocol. For immunofluorescence staining the sections were labeled with anti-ApoE, anti-factor H and anti-C5b9 antibodies. Negative controls were processed in parallel without primary antibody. Mass spectrometry analysis of protein composition in HDL and plaque samples For proteomics analysis, 20 µg of HDL and 5.8 µg of plaque samples were adjusted to a final volume of 100 µl with 100 mM NH 4 HCO 3 (Cat. No. A6141-500G, Sigma-Aldrich). Plaque samples in 8 M urea were diluted to a final concentration of 1 M urea by adjusting the volume to 100 µl with 100 mM NH 4 HCO 3 . All sample types were then reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (Cat. No.20490, Thermo Scientific), alkylated with 10 mM iodoacetamide (Cat. No. 122271000, Acros Organics) at room temperature, pH-adjusted using 1 M NH 4 HCO 3 , and digested with Sequencing Grade Modified Trypsin (Cat.no. V5113, Promega) at 37°C for 16 hours. Following digestion, the samples were acidified with 10% trifluoroacetic acid (TFA, Cat. No. 85049.051, VWR) and desalted with BioPureSPN PROTO 300 C18 Mini columns (Cat. No. HUM S18V, Nest Group) according to manufacturer’s instructions. The desalted samples were dried in a centrifuge concentrator (Concentrator Plus, Eppendorf) and the peptides were reconstituted in 30 µl buffer A, consisting of 0.1% (vol/vol) TFA, 1% (vol/vol) acetonitrile (Cat. No. 83640.320, VWR) in HPLC-grade water (Cat. No. 10505904, Fisher Scientific). For the DIA analysis, the resuspended peptides were further diluted 1:20 and 1:4, respectively, in buffer A1 (1% formic acid in HPLC-grade water). A total of 20 µl was loaded onto an Evotip (Evosep) according to manufacturer’s instructions. The desalted samples were analyzed using the Evosep One liquid chromatography system coupled to a hybrid trapped ion mobility quadrupole TOF mass spectrometer (Bruker timsTOF Pro, Bruker Daltonics) via a CaptiveSpray nano-electrospray ion source (Bruker Daltonics). Peptide separation was performed using an 8 cm × 150 µm column with 1.5 µm C18 beads (EV1109, Evosep) and the 60 samples per day method (21 min gradient time). Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. MS analysis was performed in the positive-ion mode using dia-PASEF method [18] with sample-optimized data-independent acquisition (DIA) scan parameters. To adjust DIA-PASEF parameters optimally to each sample type (HDL, plaque), data-dependent acquisition (DDA) in PASEF mode was first performed on pooled samples. The default DIA-short-gradient acquisition method was then adjusted based on the sample-specific DDA-PASEF run using the timsControl software (Bruker Daltonics). The following parameters were modified for each sample type: m/z range (405.1–1255.1); mobility range (0.85–1.30 1/K0); mean cycle time (1.80 s.). The ion mobility windows were optimized to best match the ion cloud density from the sample-type-specific DDA-runs. To analyze DIA-PASEF data, the raw (.d) files were processed using DIA-NN v18.0 [19] with a spectral library generated from the UniProt human proteome (UP000005640, downloaded 4.5.2022 as a FASTA file, 20378 proteins). The following settings were applied during library generation: fixed modifications: carbamidomethyl (C); variable modifications: acetyl (protein N-term), oxidation (M); enzyme: Trypsin/P; maximum missed cleavages: 1; mass accuracy: 1.5e-05 (MS2) and 1.5e-05 (MS1); fragment m/z: 100–1700; peptide length: 7–30; precursor m/z: 300–1600; precursor charge: to 2–4; protein inference: not performed. All other settings were set to default. Statistical analysis of the proteomics data The DIA-NN Report.pg_matrix file was used as the input to further DIA data analysis. Data pre-processing was performed using an in-house R-script. Raw intensity values were log 2 -transformed and median-normalized. Missing values were imputed using QRILC imputation. For sample group comparison, p-values were calculated using Student’s t-test with SciPy package [20] in Python. Multiple testing correction was applied using Benjamini–Hochberg method via the Statsmodels package. Volcano plots were generated using q-value threshold of 0.01 and log 2 fold-change thresholds of ± 1. Lipid mediator analysis EDTA-plasma samples (0.5 ml) were mixed with 2 ml of ice-cold methanol (LiChrosolv®, Merck) containing internal standards (500 pg each: d 8 -5-HETE, d 4 -Leukotriene B 4 , d 4 -PGE 2 , d 5 -LXA 4 , d 5 -RvD2; all from Cayman Chemical). Lipid mediators were then extracted following previously described principles [21]. Samples were precipitated at -20°C for 45 min, centrifuged at 1900xg for 10 min at 4°C, and supernatant was evaporated under nitrogen stream until reduced to 1 ml. Next, 9 ml of pH 3.5 MQ water was added, and samples were immediately loaded on Sep-Pak Vac 6 cc C18 cartridges (500 mg, Waters) in Waters Extraction Manifold. The solid phase extraction cartridges were equilibrated with 12 ml of methanol and 6 ml of MQ water. After sample loading, cartridges were washed with 4 ml of MQ water and 10 ml of hexane (LiChrosolv®, Merck). Lipid mediators were eluted with 8 ml of methyl format (Spectro Grade 98%, Fisher Scientific) and evaporated to dryness under a nitrogen stream. The dried samples were resuspended in 40 µl of methanol (Optima™, Fisher Scientific):MQ water 1:1 (vol/vol). The samples were spun at 10000xg at 4°C. The clear supernatant was transferred into a new insert. The whole sample was injected into 1290 Infinity II LC (Agilent technologies) system equipped with a Kinetex 2.6 µm C18 100 Å LC column (150 x 2.1 mm, Phenomenex) and coupled to a 6500 + QTrap mass spectrometer (AB Sciex). The flow rate was set to 0.200 ml/min, and the column temperature was maintained at 50°C. The 33-min LC run was as follows: the mobile phase (methanol/water/acetic acid of 20:80:0.01, vol/vol/vol) was ramped to 50:50:0.01 (vol/vol/vol) over 0.75 min, next to 80:20:0.01 from 3 min to 16.5 min, maintained until 21.75 min, and then ramped to 98:2:0.01 over 0.25 min. The gradient was maintained at 98:2:0.01 from 21.9 to 30 min before returning to the initial 20:80:0.01 composition. Standards were purchased from Cayman Chemical, and the MRM transitions. The data were analyzed with Sciex OS 2.0, following previously published parameters [21] including 1) peak area > 2000 counts, 2) retention time matching with an authentic reference standard, 3) minimum of 4 data points, 4) matching of at least 6 diagnostic ions to a reference standard including at least one backbone fragment from a representative subset of samples, and 5) signal-to-noise ratio > 5. Peaks with signal-to-noise ratio above 5 were included in the analysis and peaks with signal-to-noise ratio between 3–5 are marked in Table S2 as “Trace”. Statistical methods Shapiro-Wilk test was used for normality testing. Normally distributed data with n > 13 were analyzed by Pearson’s correlation analysis, while Spearman’s analysis was used for data sets without a normal distribution. For sample sizes n < 14 correlation analysis was performed using Kendall’s Tau-P. Data were adjusted to the age and sex of patients. Immunofluorescence intensities were adjusted based on the control images in Zen 3.1 (Lite) software. One-way ANOVA with Dunnett’s test was used for multiple comparisons of unequally distributed samples. Mann-Whitney U test was used for pairwise comparisons while Student’s t-test was used for sample sets with normal distribution. Partial Spearman correlation was used to assess the relationships between variables, adjusting for age and sex using the a custom Python script. Pearson's and Kendall's correlations were calculated using SPSS version 29.0.2.0 (20). RESULTS MDA modification of HDL is associated with reduced complement regulation in patients with atherosclerosis. It has been shown previously that preventing the MDA modification of HDL significantly increases plaque stability in a mouse model of atherosclerosis [22]. To better understand the role of MDA-HDL modification in humans, we analyzed MDA-HDL levels in 17 PAD patients who had undergone femoral artery endarterectomy, a surgical procedure for the removal of plaque buildup [14] (Table S1 ). We did not observe a correlation between MDA-HDL and HDL levels (Pearson’s r = 0.265, p = 0.360; Fig. 1 A), which is consistent with previous studies showing that plasma HDL levels do not necessarily predict HDL functionality, despite their association with cardiovascular disease risk [23]. Furthermore, the levels of MDA-HDL modification did not significantly correlate with markers of complement activation (Fig. S4A), suggesting that the MDA-HDL modifications may not reflect systemic inflammation levels, but rather indicate the inflammatory status of specific locations, such as arterial walls and liver, where HDL performs its primary functions. Because CFH binding to MDA-modified epitopes [7] and ApoE [8] is known to protect HDL from oxidative stress and complement attack [10, 24], we next analyzed whether levels of CFH bound to HDL (CFH-HDL) correlated with MDA-HDL levels. Western blot (WB) confirmed the presence of CFH and ApoE (Fig. 1 B and Fig. S4B) in HDLs isolated from the patients (Fig. S4B). We revealed that MDA-HDL levels negatively correlated with CFH-HDL levels measured by WB (Pearson’s r = -0.774, p = 0.001, Fig. 1 B) and by ELISA (Spearman’s ρ = -0.723, p = 0.0015, Fig. 1 C). Despite the known interaction of CFH with MDA-modified epitopes, the observed negative correlation suggests that CFH binding to HDL is not dependent only on MDA modifications. Indeed, we found a positive correlation between CFH-HDL and C3b deposits on HDL (Spearman’s ρ = 0.630, p = 0.009, Fig. 1 D). This correlation was significant in patients with ApoE-ε4 allele (Kendall’s τ = 0.511, p = 0.04) but not with non-ε4 ApoE alleles (Kendall’s τ = 0.536, p = 0.21, Fig. 1 E). Notably, no correlation was observed between MDA-HDL and C3b deposition (Pearson’s r = -0.206, p = 0.481, Fig. 1 F), suggesting that HDL particles without MDA modification are better protected by CFH under conditions of excess complement C3b, as expected in patients with atherosclerosis. CFH-mediated protection of HDL shifts HDL proteome toward cardioprotective pathways and increases cholesterol efflux. It has been shown previously that MDA modification of HDL affects its anti-inflammatory properties by altering the HDL proteome [25]. This alteration includes the enrichment of HDL proteins involved in lipid metabolism, oxidative stress, and complement activation [26]. To assess whether HDL proteomes in our PAD patients were affected by MDA modification and whether increased CFH-HDL levels promote HDL’s anti-inflammatory properties, we used mass spectrometry to compare three samples with high CFH-HDL levels to three samples with low CFH-HDL levels. Signaling pathway analysis identified several canonical pathways directly associated with vascular health (Fig. 2 A). Among significant pathways, we found: 1) Plasma lipoprotein assembly, remodeling, and clearance, 2) Liver X receptor (LXR) activation, 3) DHCR24 (24-dehydrocholesterol reductase) signaling, and 4) Atherosclerosis signaling. LXR-mediated signaling is known to play a protective role in atherosclerosis by regulating lipid metabolism, promoting reverse cholesterol transport, and suppressing inflammation in the vessel wall. Previous studies have shown that selective LXR activation in macrophages reduced atherosclerotic lesions, improved plasma lipid profiles, and reduced cholesterol accumulation in the mouse model of atherosclerosis [27]. DHCR24 has also been previously implicated in vascular health due to its involvement in cholesterol biosynthesis, oxidative stress reduction, and endothelial protection [28]. A detailed analysis of HDL proteomic signatures revealed increased expression of proteins associated with vascular health and anti-atherogenic pathways in the high CFH-HDL group (Fig. 2 B and Dataset 1). Among the proteins enriched > 2-fold, we identified AP2A2 (Log 2 fold change, Log 2 FC ≈ 5.7), a subunit of the adaptor-related protein complex 2 (AP2), which regulates macrophage cholesterol trafficking and inflammatory activity [29]. Additionally, upregulation of glutaredoxin-1 (GLRX) (log 2 FC ≈ 1.2) has been previously linked to reduced plaque burden and cardiovascular disease severity in mice [30], while RAB3D (log 2 FC ≈ 2.6) is known to be downregulated in injured carotid arteries [31]. Interestingly, two proteins significantly enriched in the high CFH-HDL group (CAMP and AP2A2) possess antimicrobial activities, supporting the long-standing hypothesis that HDL plays a role in immune defense [32, 33]. Thus, pathway analysis revealed that PAD patients with high CFH-HDL demonstrate proteome profiles associated with vascular protection, lipid metabolism, and antimicrobial activity, assuming their potential role in slowing atherosclerosis. Since our analysis of signaling pathways identified several potential mechanisms involved in cholesterol trafficking, we examined whether CFH binding to HDL helps protect HDL function by enhancing cholesterol efflux. Indeed, HDL samples from patients with high CFH-HDL levels showed enhanced cholesterol efflux compared to HDL samples from patients with low CFH-HDL levels (Fig. 2 C). To further explore the effects of CFH-mediated complement regulation of HDL, we exposed HDL to complement attack. In the absence of CFH, complement exposure led to C3b deposition on HDL (Fig. S2B) and a significant reduction of HDL-mediated cholesterol efflux (Fig. 2 D). As expected, adding CFH to the reaction decreased C3b deposition on HDL and preserved its efflux capacity. These findings align with our mass spectrometry data, suggesting that increased CFH-HDL levels enhance HDL function. Femoral plaque proteome reveals stability biomarkers linked to elevated CFH-HDL levels. Our previous results, which showed no correlation between MDA-HDL levels and plasma markers of complement activation (Fig. S4A), suggest that inflammation is localized in arterial walls near atherosclerotic plaques rather than systemically. To further investigate the relationship between circulating HDL composition and local atherosclerotic lesions in PAD patients, we used mass spectrometry to analyze proteomic profiles of femoral plaques dissected from the same group of six patients with high and low CFH-HDL levels as in HDL proteome analysis. Comparison of the plaque proteomes revealed differential expression of four proteins: HSPG2 (aka perlecan, Log 2 FC ≈ 2.2), LIMCH1 (Log 2 FC ≈ 4.5), NEGR1 (Log 2 FC ≈ 5.6), and COL18A1 (Log 2 FC ≈ 2.5) (Fig. 3 A and Dataset 2). According to the signaling pathway analysis, these proteins are involved in extracellular matrix organization and collagen biosynthesis/degradation, suggesting a role in plaque stability (Fig. 3 B and Table S3). A closer examination of the up- and downregulated proteins in each indicated pathway revealed a correlation between the protein levels and plaque stability markers (Fig. 3 C and Table S3). For example, perlecan and COL18A1 (Collagen, Type XVIII, Alpha 1), both indicative of stable plaques [34–36], were enriched in plaques of patients with high CFH-HDL levels [37]. Perlecan is a heparan sulfate proteoglycan found in the basement membrane, where it interacts with CFH [38] and PRELP (proline/arginine-rich end leucine-rich repeat protein) [39], both of which can prevent complement attack. Since our analysis of the plaque proteomes revealed elevated perlecan levels in the high CFH-HDL group, we investigated the relevance of this finding by measuring serum perlecan levels in the PAD patients. We found no correlation between MDA-HDL and serum perlecan levels (Fig. 3 D), suggesting that local perlecan concentration within plaques is more relevant than systemic perlecan levels. Perlecan is also known to reflect endothelial damage and cardiovascular complications in atherosclerosis patients with chronic kidney disease (CKD) [40]. Interestingly, CFH-HDL levels showed a suggestive correlation with platelet counts (Table S4) indicating that CFH-HDL may reduce the inflammatory load and, in turn, decrease plaque instability. Serum perlecan levels correlated positively with creatinine levels (Spearman’s ρ = 0.550, p = 0.0016, Fig. 3 E) and negatively with glomerular filtration rates (GFR) (Pearson’s r = -0.547, p = 0.003, Fig. 3 F). Since GFR and creatinine are markers of kidney function, this suggests that perlecan may serve as a possible marker for CKD in atherosclerosis patients [40]. However, serum perlecan levels did not differ between the high and low CFH-HDL groups, indicating that they reflect kidney function in atherosclerosis patients rather than plaque stability. We also observed that individuals carrying one ε4 allele in ApoE heterozygous (ApoE-ε3/ε4) had significantly higher perlecan levels than those with the ε2 or ε3 alleles (Fig. 3 G). This finding suggests that ApoE4 may increase the risk of endothelial damage, possibly due to its inability to bind CFH [10]. The role of LIMCH1 (LIM and Calponin Homology Domains 1) in atherosclerosis remains largely unexplored, but its downregulation has been observed in ruptured atherosclerotic samples from carotid arteries [41]. High CFH-HDL levels are associated with a plaque-stabilizing sheet calcification pattern. Plaque calcification develops through inflammation-driven mechanisms that influence atherosclerosis progression 54 . It varies dynamically, with nodular, sheet, and ectopic bone patterns marking disease chronicity and instability, and can be used as an independent predictor of atherosclerosis-related cardiovascular events 55 . Nodular calcification increases thrombotic risk via cap disruption; sheet calcification stabilizes plaques but increases their rigidity; ectopic bone reflects chronic inflammation while lowering rupture risk. Given the role of HDL in calcification of vascular cells, we examined calcification patterns 56 and their association with CFH-HDL and MDA-HDL levels in PAD patients (Fig. 4 ). A significant correlation was observed between CFH-HDL levels and sheet calcification (Spearman’s ρ = -0.573, p = 0.032) (Fig. 4 A). Suggestive but not significant negative association was observed between CFH-HDL levels and nodular calcification or ectopic lamellar bone (Fig. 4 B- 4 C). MDA-HDL did not correlate with either nodular/sheet calcification or lamellar bone (Fig. 4 D-F). The observed positive correlation between CFH-HDL and sheet calcification suggests a plaque-stabilizing role, potentially reducing instability and thrombotic risk in atherosclerosis. In contrast, MDA-HDL showed no significant association with sheet or nodular calcification, indicating a lesser role in plaque stability, though its correlation with bone calcification, previously linked to chronic inflammation, may reflect prolonged disease activity. Distribution of CFH-HDL, ApoE, and membrane attack complex C5b-9 in femoral plaques of PAD patients. In atherosclerosis, infiltrating macrophages in the intima take up oxidized LDL, forming foam cells that contribute to plaque buildup. HDL helps remove cholesterol from these macrophages, but its function can be impaired in diseased vessels, reducing its ability to prevent foam cell formation and inflammation. Previously, we demonstrated that CFH binding to ApoE, which is associated with HDL in human plasma, protects HDL from complement attack and thereby contributes to its anti-inflammatory properties [8]. The observed negative correlation between CFH-HDL and MDA-HDL in PAD patients (Fig. 1 B-C), along with prior findings linking MDA-HDL inhibition to plaque stability [22], led us to examine the distribution of CFH-HDL, ApoE, and the membrane attack complex C5b-9 in femoral plaques. Cryosections of femoral plaques from three patients with high CFH-HDL levels and three patients with low CFH-HDL levels were stained with hematoxylin-eosin (H&E) (Fig. 5 and Fig. S5), while adjacent sections were stained with antibodies against CFH, ApoE, and C5b-9 (Fig. 5 and Fig. S3). Parallel histological and immunohistochemical analyses revealed that CFH and ApoE were primarily colocalized in plaques from high CFH-HDL patients, particularly at the periphery of fibrotic-necrotic plaque cores and in pericytic regions (Fig. 5 B). Morphological assessment combined with DAPI nuclear counterstaining confirmed cellular association of the proteins (Fig. 5 B and Fig. S3). Notably, C5b-9 deposition exhibited spatial correlation with CFH-HDL and ApoE in high CFH-HDL plaques, providing direct histological evidence that CFH-ApoE interaction modulates localized complement activation within atherosclerotic lesions. These findings align with established mechanisms, whereby CFH binding to HDL-associated ApoE enhances complement regulation while promoting cholesterol efflux from macrophage foam cells [24]. Synergistic effect of CFH-HDL and specialized pro-resolving mediators on vascular inflammation in atherosclerosis patients Pro-inflammatory mediators contribute to ongoing inflammation in atherosclerotic plaques, promoting plaque instability and increasing the risk of cardiovascular events [11, 42]. In contrast, specialized pro-resolving mediators (SPMs), play a crucial role in resolving inflammation, stabilizing plaques and preventing rupture (Fig. 6 A). To explore the potential synergistic interaction between SPMs and CFH-HDL in resolving inflammation in atherosclerotic plaques, we measured circulating lipid mediator levels in PAD patients using mass spectrometry, and assessed their correlation with previously determined biochemical markers and plaque calcification patterns (Fig. 6 B). CFH-HDL levels positively correlated with plasma levels of SPM 15-epi-LXA 4 (Spearman’s ρ = 0.800, p = 0.0006, Fig. 6 C) and its precursor 15-HETE (Spearman’s ρ = 0.574, p = 0.025) (Fig. 6 D). The levels of lipid mediators and their pathway markers did not correlate with the MDA-HDL levels, although a trend to an inverse association was observed for the 15-HETE pathway mediators (sum of lipoxins (LX) and 15-HETE) (Fig. 6 E). Further analysis showed that sheet calcification, exhibited a positive correlation with 15-HETE (Spearman’s ρ = 0.596, p = 0.025), and 17-HDHA (Spearman’s ρ = 0.661, p = 0.010), (Fig. 6 F-G). Moreover, the potent anaphylatoxin, C5a, correlated positively with PGD2 (Spearman’s ρ = 0.741, p = 0.0016) (Fig. 6 H). While several of these lipid mediators have protective effects in atherosclerosis [43] or are metabolites of SPM pathways (Fig. 6 A), PGD2 has a dual role as a pro-resolving and pro-inflammatory mediator. Thus, these data suggest that the general increase in pro-resolving lipid mediators and the presence of HDL-associated CFH refers to a more responsive regulation of inflammation. These findings suggest that CFH-HDL levels may influence both the distribution patterns and extent of atherosclerosis in PAD patients. However, given the small sample size, these observations warrant further investigation with larger cohorts to establish their significance. To sum up, our data suggest a synergy between CFH-HDL and SPMs as anti-inflammatory and anti-atherogenic molecules, which may serve as a potential target for inhibiting plaque instability. Along with other identified biomarkers, these factors could potentially be used for diagnosing individuals with high-risk, rupture-prone atherosclerotic plaques. DISCUSSION Atherosclerotic plaque structure significantly affects its susceptibility to rupture, and therefore, understanding the key factors determining the stability of the plaque is essential. Our analysis of the oxidative modification, the protein profile of circulating HDL, and plasma metabololipidome, shows that the circulating molecular profiles markedly mirror plaque histomorphology, and their inflammatory microenvironment. HDL has a targeted function on the artery wall and the potential to prevent the development of atherosclerosis. We analyzed the presence of MDA adducts and CFH levels in circulating HDL of PAD patients and discovered immune molecules and structural components of plaques (Fig. 7 ) strongly implying human plaque instability, as evidenced by the plaque proteome, histological features, and metabololipidome. The observations that high levels of plasma lipid mediators and their pathway markers were associated with increased CFH-HDL levels, plasma complement activation markers C3a and C5a, and sheet calcification in plaques are novel. Apparently, the complement cues of inflammation and counteracting responses effectively enhance the SPM production in certain patients to attenuate the inflammation [43]. Since the levels of the markers of SPM pathways 15-HETE and 17-HDHA showed an inverse trend with MDA-HDL, the SPMs may also help mitigate oxidative stress [45]. These observations highlight the pathophysiological role of active lipid mediator biosynthesis in promoting plaque stability through their pro-resolving functions. The major histological markers for plaque instability include atherosclerotic lesion collagen content, fibrous cap thickness, and nodular calcification. Previous studies indicate that calcified nodules and superficial calcifications correlate with plaque vulnerability, whereas substantial and widespread calcifications promote plaque stabilization [46]. Transcriptomic studies show that highly calcified, stable plaques are associated with the upregulation of smooth muscle-related pathways and downregulation of inflammation. Previous proteomic and transcriptomic analyses of arterial plaques in coronary artery disease and carotid artery disease have identified several factors implying plaque vulnerability including extracellular matrix proteins, genetic markers, and inflammatory macrophages [35] [47]. Although some of these proteins/pathways have been implicated before, our observation that these immune molecules and structural components of plaques significantly associate with patient CFH-HDL levels is novel and thereby highlighting important roles for complement regulation in atherosclerosis. Furthermore, we provide mechanistic rationale (Fig. 2 C-D) for the anti-atherogenic and anti-inflammatory functions of HDL-associated CFH. In atherosclerosis, MDA-HDL epitopes are important mediators for inflammation and potential targets for therapeutic interventions. On atherosclerotic plaques, however, CFH binds to MDA-LDL and thereby reduces the pro-inflammatory effects of MDA in a murine model [7]. Interestingly, we observed that in ApoE4-carriers increased C3b depositions on HDL resulted in a corresponding increase in CFH levels. These increased CFH-HDL levels correlated positively with the pro-resolving 15-epi-LXA 4 and its pathway marker 15-HETE. These mediators promote resolution by blocking neutrophil chemotaxis and transepithelial migration, decreasing the NF-κB signaling and production of pro-inflammatory cytokines, and, importantly, increasing efferocytosis, the clearance of apoptotic cells [48]. These findings suggest that CFH-HDL may help protect against the disease by counteracting ongoing inflammation. Furthermore, CFH has the capacity to facilitate clearance of cellular debris which is characteristic in this disease. The crucial role of CFH in the non-inflammatory clearance is exemplified by the Y402H polymorphism in CFH, where impaired complement regulation leads to metabolic debris and drusen accumulation in age-related macular degeneration (AMD) [10]. Moreover, CFH’s inability to bind the Alzheimer’s disease-associated ApoE4 increases Aβ oligomerization, promoting plaque formation and neuroinflammation in Alzheimer’s disease [10]. The importance of CFH in regulating complement in various tissues can be attributed to its soluble nature, allowing it to reach areas where complement activation is exacerbated, such as atherosclerotic plaques. Here we provide further evidence of its role in protection against complement in atherosclerotic plaques ex vivo , as evidenced by the immunofluorescence microscopy demonstrating colocalization between ApoE, CFH, and C5b-9 (Fig. 4 ). Additionally, the suggestive negative correlation between patient CFH-HDL levels and platelet counts indicates that reduced CFH levels may promote chronic inflammation and platelet activation, both of which are known to increase the risk of cardiovascular events in atherosclerosis patients [49]. Furthermore, the protective role of CFH in atherosclerosis is supported by evidence that CFH V62I polymorphism correlates with the serum levels of matrix metalloproteinase 8 (MMP-8), which is a pro-inflammatory enzyme linked to cardiovascular diseases [50]. The strengths of our study include a novel approach to identifying a comprehensive set of immune molecules and structural components of plaques through high-throughput mass spectrometry methods covering the proteome and lipidome of patient HDLs, serum and atherosclerotic plaques, and metabololipidome of plasma. Our study has limitations. First, ultracentrifugation reduces the detection of low-affinity proteins that could be crucial to HDL’s function. In addition, we did not differentiate between HDL subpopulations that may differ in their anti-atherogenic functions. Second, our study population of 17 patients is small, and verification of these results requires studies with larger patient cohorts. Furthermore, the patients studied had advanced atherosclerotic disease requiring invasive treatment. Additional work is required to determine the pathways by which complement regulation and pro-resolving responses are linked. CONCLUSIONS By using advanced proteomic and metabololipidomic profiling of patient HDL, plasma, and femoral artery plaques, this study provides molecular insights into the inflammatory mechanisms underlying plaque rupture. Importantly, CFH-HDL concentrations correlated positively with both pro-resolving lipid-mediator pathways and increased extracellular matrix proteins, while inverse patterns were linked to plaque instability. Furthermore, we demonstrated that CFH on HDL increased the capacity of HDL to promote cholesterol efflux, providing direct mechanistic evidence for CFH-HDL as an anti-atherogenic molecule. These findings suggest that complement regulation by CFH and the pro-resolving mechanisms of SPMs work synergistically to promote plaque stability and counteract extracellular matrix breakdown. Our results underscore the potential of circulating biomarkers to reflect plaque pathology, offering clinically relevant insights into mechanisms of plaque stability and pointing to new therapeutic strategies for managing atherosclerotic disease. Abbreviations CFH Complement factor H HDL High-density lipoprotein MDH malondialdehyde PAD peripheral arterial disease HDL-C HDL-cholesterol LDL low-density lipoprotein ApoE apolipoprotein E SPMs specialized pro-resolving mediators BSA bovine serum albumin HRP Horseradish peroxidase PBS Phosphate-buffered saline TMB 3,3',5,5'-Tetramethyl- - benzidine ELISA enzyme-linked immunosorbent assay EDTA Ethylenediaminetetraacetic acid HSPG heparan sulpfate proteoglycan PMA Phorbol 12-myristate 13-acetate H&E Hematoxylin and Eosin TFA Trifluoroacetic acid DIA data-independent acquisition DDA data-dependent acquisition PASEF parallel accumulation–serial fragmentation QRLIC Quantile regression approach for left-censored missing HETE Hydroxyeicosatetraenoic acid PGE Prostaglandin E2 LXA A lipoxin RvD2 Resolvin D2 MQ Milli Q LXR Liver X receptor DHCR24 24-dehydrocholesterol reductase AP2A2 AP-2 complex subunit alpha-2 AP2 adaptor-related protein complex 2 GLRX glutaredoxin-1 RAB3D Ras-related protein CAMP Cyclic adenosine monophosphate LIMCH1 LIM and calponin homology domains-containing protein 1 NEGR1 Neuronal growth regulator 1 COL18A1 collagen type XVIII alpha 1 chain PRELP proline/arginine-rich end leucine-rich repeat protein CKD chronic kidney disease GFR glomerular filtration rate 15-epi-LXA 4 15-epi-lipoxin A4 DHA Docosahexaenoic acid MRA magnetic resonance angiogram MMP-8 matrix metalloproteinase 8 Declarations Ethics approval and consent to participate and consent for publication The study protocol was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa (ASO‐project 78/13/03/00/2014), and all procedures were conducted in accordance with applicable guidelines and regulations.Informed consent for publication has been obtained from participants or their legal guardians. Authors' contributions Conceptualization: KH, EN, SS, PU Methodology: EN, SS, PU, IL, ML, LC, ER, EV, AT, SK, CM, MH Investigation: KH, EN, SS, PU, IL, ML, LC, ER, EV, AT, SK, CM, MM, MH, RK, JS Visualization: KH, EN, SS, PU, IL, ML, CM, MH, JS Funding acquisition: KH Project administration: KH Supervision: KH, MV, JS, RK Writing – original draft: KH, EN, SS, PU Writing – review & editing: KH, EN, SS, PU, IL, ML, LC, ER, EV, AT, SK, CM, MM, SM, MH, MV, JS, RK Funding: The Finnish foundation for cardiovascular research (190044 and 230023) Academy of Finland (1331108) Jane and Aatos Erkko foundation (190002) Ida Montin foundation (2021) Biomedicum Helsinki foundation (20220141). Acknowledgments Dr. Matti Jauhiainen (Minerva Institute, Helsinki, Finland) for reviewing the manuscript and providing the rabbit anti-apoE antibody. Dr. Anastasia Ludwig (Neuroscience Center, HiLIFE, Helsinki, Finland) for providing the Python script for statistical analyses. Lauri Snellman, Vellamo Tuomainen and Juuso Vedenjuoksu for their excellent assistance in the laboratory. Infrastructures Bioimaging unit (BIU), University of Helsinki, Finland. Competing interests: Authors declare that they have no competing interests. Data and materials availability: Data are available in the main text or the supplementary materials. Proteomic data files will be uploaded in MassIVe with ID numbers. References Aday AW, Everett BM: Dyslipidemia Profiles in Patients with Peripheral Artery Disease . Curr Cardiol Rep 2019, 21 (6):42. Syed S, Nissila E, Ruhanen H, Fudo S, Gaytan MO, Sihvo SP, Lorey MB, Metso J, Oorni K, King SJ et al : Streptococcus pneumoniae pneumolysin and neuraminidase A convert high-density lipoproteins into pro-atherogenic particles . iScience 2021, 24 (6):102535. 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Circ Cardiovasc Genet 2017, 10 (6). Additional Declarations No competing interests reported. Supplementary Files BMCsuplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6828685","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472565005,"identity":"6266e9e6-1274-43ec-9b3f-a2e02c0807b4","order_by":0,"name":"Eija Nissilä","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Eija","middleName":"","lastName":"Nissilä","suffix":""},{"id":472565006,"identity":"37d89484-6458-485d-889c-7e125a2044e1","order_by":1,"name":"Shahan Syed","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Shahan","middleName":"","lastName":"Syed","suffix":""},{"id":472565007,"identity":"70a58fb4-19d0-4a0c-aa02-4e68bf044516","order_by":2,"name":"Pavel Uvarov","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Uvarov","suffix":""},{"id":472565009,"identity":"6385f7ea-0172-496d-ba4c-fe406b21538e","order_by":3,"name":"A. 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Mäyränpää","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Mikko","middleName":"I.","lastName":"Mäyränpää","suffix":""},{"id":472565018,"identity":"3dad2941-6244-40d0-bfdc-2a03b4788429","order_by":12,"name":"Seppo Meri","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Seppo","middleName":"","lastName":"Meri","suffix":""},{"id":472565019,"identity":"b3540afe-77ee-4ec5-97cd-c269bd03bf47","order_by":13,"name":"Minna Holopainen","email":"","orcid":"","institution":"Helsinki University Lipidomics Unit (HiLIPID), Helsinki Institute of Life Science (HiLIFE), University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Minna","middleName":"","lastName":"Holopainen","suffix":""},{"id":472565020,"identity":"ba8b85ab-eca8-4581-afb4-1de994429ffe","order_by":14,"name":"Reijo Käkelä","email":"","orcid":"","institution":"Helsinki University Lipidomics Unit (HiLIPID), Helsinki Institute of Life Science (HiLIFE), University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Reijo","middleName":"","lastName":"Käkelä","suffix":""},{"id":472565021,"identity":"18bb71d0-c960-4313-bc4f-72290116dfce","order_by":15,"name":"Markku Varjosalo","email":"","orcid":"","institution":"HiLIFE Helsinki Institute of Life Science University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Markku","middleName":"","lastName":"Varjosalo","suffix":""},{"id":472565022,"identity":"872c5322-09ec-4ba7-9768-be6760402893","order_by":16,"name":"Juha Sinisalo","email":"","orcid":"","institution":"Helsinki University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Juha","middleName":"","lastName":"Sinisalo","suffix":""},{"id":472565023,"identity":"ff3154fd-079b-4540-b90a-63bb0e4fe32e","order_by":17,"name":"Karita Haapasalo","email":"data:image/png;base64,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","orcid":"","institution":"University of Helsinki","correspondingAuthor":true,"prefix":"","firstName":"Karita","middleName":"","lastName":"Haapasalo","suffix":""}],"badges":[],"createdAt":"2025-06-05 11:23:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6828685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6828685/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85363873,"identity":"13e83c87-a7bf-4a01-8b9f-acbfc1aa08d6","added_by":"auto","created_at":"2025-06-25 06:25:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation between MDA-HDL and CFH-HDL levels in atherosclerosis patients. A\u003c/strong\u003e) No correlation was observed between MDA-HDL and HDL levels in PAD patients (n=16). \u003cstrong\u003eB\u003c/strong\u003e) WB detected CFH and ApoE products in the HDL samples from PAD patients (n=17) and controls. An inverse correlation between MDA-HDL and CFH-HDL levels was revealed by WB. \u003cstrong\u003eC)\u003c/strong\u003e ELISA, showed an inverse correlation between MDA-HDL and CFH-HDL levels (n=17). \u003cstrong\u003eD\u003c/strong\u003e) The observed correlation between the levels of CFH-HDL and C3b deposition on HDL \u003cstrong\u003eE\u003c/strong\u003e) was significant among PAD patients expressing the ApoE4 isoform (squares, n=9), but not among patients lacking the ApoE4 allele (circles, n=7). \u003cstrong\u003eF\u003c/strong\u003e) No correlation was found between the levels of MDA-HDL and C3b deposition on HDL. The average values are from three independent experiments, each performed with two technical duplicates, except for C3b deposition data (n=2). Correlation coefficients (\u003cem\u003er\u003c/em\u003e), (\u003cem\u003eρ\u003c/em\u003e), and (\u003cem\u003eτ\u003c/em\u003e) as well as corresponding p-values were adjusted for the age and sex of patients.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/ff29f5036fac71c3d539c337.png"},{"id":85363871,"identity":"4cf2de21-c181-4008-afb3-c35762973694","added_by":"auto","created_at":"2025-06-25 06:25:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":598917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMass spectrometry analysis of HDL proteomes identifies markers associated with high CFH-HDL levels that promote HDL-mediated cholesterol efflux in atherosclerosis patients.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) Ingenuity canonical and disease pathways associated with differentially expressed (DE) proteins in HDL from high vs. low CFH-HDL patient groups (n=6). \u003cstrong\u003eB\u003c/strong\u003e) DE proteins showing significant changes (p \u0026lt; 0.05) indicated by an asterisk. \u003cstrong\u003eC-D)\u003c/strong\u003e Cholesterol efflux of HDL controls (5 and 10 µg), patient HDLs with high and low CFH-HDL levels (n=6), as well as \u003cstrong\u003e(D)\u003c/strong\u003e HDL treated with buffer, complement C3, factor B, and factor D (C3+B+D) or complement C3, factor B, factor D, and CFH (C3+B+D+CFH).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/3de8ef9cc7a4d8f2fbc5d95a.png"},{"id":85362172,"identity":"fe2a2f1b-03aa-4523-9c05-6704727dfa6b","added_by":"auto","created_at":"2025-06-25 06:17:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":831323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMass spectrometry analysis of atherosclerotic plaque proteomes identifies markers and signaling pathways associated with high CFH-HDL levels. A)\u003c/strong\u003e Volcano plot highlighting significantly enriched proteins in plaques from patients with high vs. low CFH-HDL levels (n=6) \u003cstrong\u003eB)\u003c/strong\u003e and the canonical pathways associated with DE proteins between the groups. \u003cstrong\u003eC)\u003c/strong\u003e DE proteins showing significant changes (p \u0026lt; 0.05) indicated by asterisks. \u003cstrong\u003eD)\u003c/strong\u003e No correlation was found between serum perlecan levels and MDA-HDL levels. \u003cstrong\u003eE)\u003c/strong\u003e Perlecan levels correlated positively with serum creatinine levels \u003cstrong\u003eF)\u003c/strong\u003e and negatively with glomerular filtration rates (GFR). \u003cstrong\u003eG)\u003c/strong\u003eIncreased serum perlecan levels were observed in ApoE heterozygous patients. The average values are based on three independent experiments with two technical replicates. Correlation coefficients (\u003cem\u003er\u003c/em\u003e) and (\u003cem\u003eρ\u003c/em\u003e) as well as corresponding p-values were adjusted for the age and sex of patients.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/b10171a618a79bb785eae9cf.png"},{"id":85362177,"identity":"1ad84025-23ef-4a85-a394-854d0dc819e3","added_by":"auto","created_at":"2025-06-25 06:17:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis of\u003c/strong\u003e \u003cstrong\u003eCFH-HDL and MDA-HDL levels with calcification patterns in atherosclerotic plaques.\u003c/strong\u003e \u003cstrong\u003eA-C)\u003c/strong\u003eCFH-HDL levels showed a significant correlation with sheet calcification but no correlation with nodular calcification, or ectopic lamellar bone area \u003cstrong\u003eD-F)\u003c/strong\u003e MDA-HDL levels did not correlate significantly with any of the calcification patterns in atherosclerotic plaques. Average values were derived from three independent experiments, each performed with two technical duplicates. Data following a normal distribution were analyzed using Pearson’s correlation, while Spearman’s analysis was used as a non-parametric alternative. Correlation coefficients (\u003cem\u003er\u003c/em\u003e) and (\u003cem\u003eρ\u003c/em\u003e) as well as corresponding p-values were adjusted for age and sex of patients.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/fac7caec408a3a07210c14fc.png"},{"id":85362180,"identity":"826ee9ac-63ea-45c2-a058-55d07c9dda8f","added_by":"auto","created_at":"2025-06-25 06:17:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":680062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological and immunofluorescence analysis\u003c/strong\u003e \u003cstrong\u003eof atherosclerosis plaques from patients with high and low CFH-HDL levels.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Representative H\u0026amp;E-stained images of femoral plaques from two PAD patients with high (patients 18 and 89) and low CFH-HDL levels (patient 19). (See Fig. S5). \u003cstrong\u003eB)\u003c/strong\u003e Immunofluorescence analysis reveals co-localization of ApoE, CFH, and C5b-9 around cells and at plaque edges, suggesting complement regulation. Fluorescence intensities were lower in the low CFH-HDL sample (patient 19) compared to high CFH-HDL samples. Negative control sample (without primary antibody). ApoE genotype and Scale bar: 500 µm (H\u0026amp;E full sections) are indicated. Magnified areas from (A) are shown in (B).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/6561b857d8e9825705ddaef8.png"},{"id":85362170,"identity":"9ae3699b-358a-4760-aee1-259c801161b8","added_by":"auto","created_at":"2025-06-25 06:17:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":687557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations of lipid mediator pathways with CFH-HDL, MDA-HDL, plaque stability, and complement activity. A\u003c/strong\u003e. Simplified schematic presentation of the lipid mediator pathways that either produce PGD\u003csub\u003e2\u003c/sub\u003e or have 5-HETE, 15-HETE or 17-HDHA metabolites. Ovals represent intermediates and markers of the pathways. \u003cstrong\u003eB.\u003c/strong\u003e Heat map of metabololipidomics showing correlation coefficients and p-values between the mediators and MDA-HDL, CFH-HDL, C5a, and plaque calcifications. 15-HETE pathway mediators is a sum of 15-epi-LXA4, LXB4, 5S,15S-diHETE and 15-HETE, and 17-HDHA pathway mediators is a sum of RvD5, PD1, PDX and 17-HDHA) (n = 16). Specific lipid mediators or pathway markers correlated statistically significantly with \u003cstrong\u003eC.\u003c/strong\u003e CFH-HDL, \u003cstrong\u003eF-G.\u003c/strong\u003e sheet calcification (n = 14) and \u003cstrong\u003eH.\u003c/strong\u003e C5a levels. Data sets were analyzed by Spearman’s correlation analysis. The correlation coefficient (ρ) and p-values are indicated.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/304afce9daffcbef9f4ef00a.png"},{"id":85363876,"identity":"3403101a-00ae-4889-b6ec-94a721775003","added_by":"auto","created_at":"2025-06-25 06:25:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":408883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA summary of the observed inflammatory and anti-inflammatory molecules and structural components of PAD plaques.\u003c/strong\u003e Subjects with stable plaques demonstrate low levels of MDA-HDL modifications along with high levels of CFH-HDL and HDL-associated AP2A2, while these parameters are inversed in the unstable plaques. CFH protects HDL from C3b deposition and MDA modifications that convert functional HDL into an attenuated form incapable of cholesterol efflux. Responsive pro-resolving lipid mediator pathways induce anti-atherogenic effects. The damaged plaque itself is characterized by deficiency in the key extracellular matrix proteins (HSPG2, LIMCH1, NEGR1 and COL18A1) and nodular calcification that indicate plaque instability. While HSPG2 (perlecan) levels are reduced in the plaques the serum perlecan concentrations are increased in patients carrying ApoE4 suggesting that reduced anti-inflammatory and plaque stabilizing functions by CFH-HDL and SPMs promote extracellular matrix breakdown. The total risk score shows the cumulative burden of the detected risk factors (red arrows).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/b3b5de50ff4c2b2bdd5222a6.png"},{"id":95526548,"identity":"b97d70ed-6564-4355-b20d-8ffadd0a8fb0","added_by":"auto","created_at":"2025-11-10 10:07:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7467089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/ac986b5e-4866-4050-902f-f1a29d4b45a3.pdf"},{"id":85362176,"identity":"4ea1c41b-5f78-4b0c-9e3b-465bcbf1212c","added_by":"auto","created_at":"2025-06-25 06:17:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4622143,"visible":true,"origin":"","legend":"","description":"","filename":"BMCsuplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6828685/v1/8c8772de2c5e27cd14fed834.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Complement Factor H and Pro-Resolving Mediators Synergistically Enhance Plaque Stability in Peripheral Arterial Disease","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAtherosclerosis, the leading cause of cardiovascular disease, is characterized by the thickening of the arterial wall, which leads to constricted blood flow. It typically co-exists in several arterial beds, and (PAD) increases the risk of coronary artery disease. The immune response and inflammation play crucial roles in the development and progression of atherosclerosis. It can become life-threatening by causing ischemia in affected organs due to restricted blood flow or by triggering plaque rupture, which releases atherosclerotic particles into the bloodstream and raises the risk of a critical thrombus. In either case, early detection is crucial for timely and effective treatment.\u003c/p\u003e \u003cp\u003eHigh-density lipoprotein (HDL) removes cholesterol from the periphery to the liver and promotes macrophage cholesterol efflux. It also has many beneficial anti-inflammatory and anti-oxidative functions that can mitigate the risk factors of cardiovascular diseases. Low levels of HDL-cholesterol (HDL-C) are recognized as a risk factor for PAD [1]. However, cardiovascular health is influenced not only by the quantity of HDL but also by its quality and functionality, which are determined by HDL size, structure, and protein and lipid composition. In addition, various functional capacities of HDL, such as antioxidant, anti-inflammatory, and cholesterol efflux-promoting activities, play a crucial role in determining its impact on vascular health. These properties can be impaired by inflammatory molecules released during metabolic disorders, atherosclerosis, and acute infections [2]. Importantly, the reduced capacity of HDL to promote cholesterol efflux from donor cells correlates with atherosclerotic burden in the coronary and carotid arteries [3]. Apolipoprotein A1 is the major determinant of HDL functionality and its formulation has been evaluated in clinical trials for the reduction of myocardial infarction, stroke, or death from cardiovascular causes. These studies, however, have not led to clinical success [4].\u003c/p\u003e \u003cp\u003eMalondialdehyde-modified high-density lipoproteins (MDA-HDLs) are formed during lipid peroxidation, and they are known to promote inflammatory responses that play a major role in the development of atherosclerosis. Even though the role of MDA-HDL in PAD has thus far not been explored, MDA-modified low-density lipoprotein (MDA-LDL) has been identified as a marker for plaque vulnerability in peripheral artery disease patients [5]. Complement activation is a key driver of inflammation and atherogenesis [6]. This process is regulated by the main fluid phase complement regulator, complement factor H (CFH), which binds to C3b molecules deposited on self-structures and to MDA-modified epitopes in atherosclerotic lesions [7]. On HDL, CFH interacts with apolipoprotein E (ApoE) to confer protection against the complement attack [8]. ApoE4 isoform is strongly associated with increased risk for Alzheimer\u0026rsquo;s disease and to a lesser extent with coronary heart disease [9]. This isoform has also been shown to confer reduced binding to CFH leading to attenuated complement regulation and increased inflammation [10].\u003c/p\u003e \u003cp\u003eIn addition to complement activation, pro-inflammatory lipid mediators are known to promote inflammation in atherosclerotic lesions, while (SPMs) attenuate and help resolve inflammation. Fatty acid-derived SPMs not only counter-regulate inflammation but also increase efferocytosis, reduce aortic plaque size and necrosis, and increase the thickness of the fibrous cap of plaques [11, 12]. Pro-inflammatory conditions often lead to increased lipid mediator production, as evidenced by elevated circulating pro-inflammatory mediator and SPM levels observed, for example, immediately after myocardial infarction. However, imbalance among different types of lipid mediators may impair plaque stability, with low SPMs increasing the risk of cardiovascular events [13].\u003c/p\u003e \u003cp\u003eIn the current study, we explored pro- and anti-inflammatory molecules in a cohort of PAD patients. Our biochemical data, including HDL oxidation and complement regulation, along with proteomics data, identified novel lipid-specific markers of HDL and plasma metabololipidome that contribute to anti-inflammatory and anti-atherogenic properties of HDL. Our histological and proteomics data showed that increased levels of these markers were associated with stability markers in femoral artery plaques and a lower artery stenosis grade. Furthermore, the histomorphological features of femoral artery plaques and observations from magnetic resonance angiography correlated with our biochemical findings. Our data suggest that complement regulation and specialized pro-resolving mediators enhance plaque stability by promoting HDL function and pro-resolving responses. These findings provide a potential target for drug development and improved disease diagnostics.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient samples and data\u003c/h2\u003e \u003cp\u003eThis study includes a subpopulation of atherosclerosis patient cohort that has been described earlier (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [14, 15]. Femoral plaque samples (n\u0026thinsp;=\u0026thinsp;6) were collected during endarterectomy of the femoral artery bifurcation and stored at -80\u0026deg;C. Magnetic resonance angiography (MRA) was used to visualize arterial flow patterns, complemented by histological examination of atherosclerotic plaques using H\u0026amp;E-stained paraffin sections to reveal the distinct patterns of the arterial disease (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The patients provided written informed consent for participation in the study prior to undergoing endarterectomy surgery. The study protocol was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa (ASO-project 78/13/03/00/2014), and all procedures were conducted in accordance with applicable guidelines and regulations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eApoE genotyping\u003c/h3\u003e\n\u003cp\u003eFour microliters of patient DNA (20 ng/\u0026micro;l) extracted from peripheral blood mononuclear cells, were used for sequencing. Previously published primers were used [16] along with with OneTaq Hot Start 2X Master Mix with Standard Buffer (New England Biolabs, Ipswich, MA, USA). The PCR reaction was performed at 98\u0026deg; C for 4 min, followed by 35 cycles of 98\u0026deg; C for 10 sec, 60\u0026deg; C for 30 sec, and 72\u0026deg; C for 40 sec and 72\u0026deg; C for 10 min. PCR products were purified from agarose gels using ExoSAP-IT\u0026trade; (Thermo Fisher) by incubating at 37\u0026deg; C for 30 min followed by 80\u0026deg; C for 15 min. The products were sequenced twice using forward and reverse primers, electrophoresis was performed using ABI3730xl DNA Analyzer, and base calling was conducted with Sequencing Analysis 7 at Finnish institute for Molecular Medicine (FIMM, University of Helsinki). The sequences were analyzed using Sequencher 4.8 software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasuring malondialdehyde (MDA) adducts on HDL.\u003c/b\u003e HDL samples were isolated from the EDTA plasma obtained from the patients and healthy volunteers [17]. MDA-HDL was measured using Oxidized HDL Assay Kit (Cat.no ab242308, Abcam) from 1:50 diluted precipitated serum samples (n\u0026thinsp;=\u0026thinsp;17). Absorbances were measured at 450 nm using Hidex Sense microplate reader (Hidex).\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eHDL (n\u0026thinsp;=\u0026thinsp;17) was coated onto 96-well microplates at a concentration of 5\u0026ndash;20 \u0026micro;g/ml in PBS (Nunc PolySorp, Cat. No. 444865) and incubated overnight at 4 \u0026deg;C. Plates were washed with PBS and blocked with 3% fatty acid-free bovine serum albumin (BSA; Cat.no. P6156, Biowest) in PBS. After washing wells were incubated with goat anti-factor H (1:2000, Cat. No. 341276, Calbiochem), rabbit anti-apoE (1:10000, a kind gift from Dr. Matti Jauhiainen), or rabbit anti-C3c (1:500, Cat. No. OSAP14/15, Behring) antibodies in 0.3% BSA-PBS for 1 hour at +\u0026thinsp;37 \u0026deg;C. After washing the wells were incubated for 40 minutes at 37 \u0026deg;C with HRP-conjugated anti-goat (1:2000, Cat. No. 705-035-147, Jackson ImmunoResearch laboratories) or anti-rabbit IgG (Cat. No. NEF812, Perkin Elmer) in 0.3% BSA/PBS. After three washes, the wells were incubated with 1-Step\u0026trade; Ultra TMB-ELISA Substrate Solution (Cat. No. 34028, Thermo Fisher Scientific). The reaction was stopped with 0.5 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and absorbance was measured at 450 nm. All experiments were conducted in triplicate or duplicate. Perlecan levels were analyzed from 1:10 or 1:20 diluted EDTA (n\u0026thinsp;=\u0026thinsp;17) or serum samples (n\u0026thinsp;=\u0026thinsp;35) using a human HSPG (perlecan) ELISA kit (Cat.no. ab274393, Abcam) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eWestern blot and cholesterol efflux assay\u003c/h3\u003e\n\u003cp\u003eThe presence of CFH, ApoE, ApoAI or C3b on HDL was determined by Western blotting (Fig. S2). HDL-2 particles (2.8 mg/ml) were incubated with complement proteins and subjected to size-exclusion chromatography and the protein concentrations were measured prior the cholesterol efflux assay. The cholesterol efflux assay was performed according to manufacturer\u0026rsquo;s instructions (Cat. No. ab196985, Abcam) using THP-1 cells activated with 100nM PMA for 72 h. The cells were incubated with 5\u0026ndash;10 \u0026micro;g of HDL. Replicate assays were normalized by averaging the values of all samples within each assay replicate and then multiplying by the calculated ratios. Each experiment was performed in at least duplicate and repeated three times.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining of peripheral artery plaques\u003c/h3\u003e\n\u003cp\u003eCryosections (Fig. S3) of femoral plaques were stained using a standard H\u0026amp;E protocol. For immunofluorescence staining the sections were labeled with anti-ApoE, anti-factor H and anti-C5b9 antibodies. Negative controls were processed in parallel without primary antibody.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry analysis of protein composition in HDL and plaque samples\u003c/h2\u003e \u003cp\u003eFor proteomics analysis, 20 \u0026micro;g of HDL and 5.8 \u0026micro;g of plaque samples were adjusted to a final volume of 100 \u0026micro;l with 100 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e (Cat. No. A6141-500G, Sigma-Aldrich). Plaque samples in 8 M urea were diluted to a final concentration of 1 M urea by adjusting the volume to 100 \u0026micro;l with 100 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e. All sample types were then reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (Cat. No.20490, Thermo Scientific), alkylated with 10 mM iodoacetamide (Cat. No. 122271000, Acros Organics) at room temperature, pH-adjusted using 1 M NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e, and digested with Sequencing Grade Modified Trypsin (Cat.no. V5113, Promega) at 37\u0026deg;C for 16 hours. Following digestion, the samples were acidified with 10% trifluoroacetic acid (TFA, Cat. No. 85049.051, VWR) and desalted with BioPureSPN PROTO 300 C18 Mini columns (Cat. No. HUM S18V, Nest Group) according to manufacturer\u0026rsquo;s instructions. The desalted samples were dried in a centrifuge concentrator (Concentrator Plus, Eppendorf) and the peptides were reconstituted in 30 \u0026micro;l buffer A, consisting of 0.1% (vol/vol) TFA, 1% (vol/vol) acetonitrile (Cat. No. 83640.320, VWR) in HPLC-grade water (Cat. No. 10505904, Fisher Scientific).\u003c/p\u003e \u003cp\u003eFor the DIA analysis, the resuspended peptides were further diluted 1:20 and 1:4, respectively, in buffer A1 (1% formic acid in HPLC-grade water). A total of 20 \u0026micro;l was loaded onto an Evotip (Evosep) according to manufacturer\u0026rsquo;s instructions. The desalted samples were analyzed using the Evosep One liquid chromatography system coupled to a hybrid trapped ion mobility quadrupole TOF mass spectrometer (Bruker timsTOF Pro, Bruker Daltonics) via a CaptiveSpray nano-electrospray ion source (Bruker Daltonics). Peptide separation was performed using an 8 cm \u0026times; 150 \u0026micro;m column with 1.5 \u0026micro;m C18 beads (EV1109, Evosep) and the 60 samples per day method (21 min gradient time). Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. MS analysis was performed in the positive-ion mode using dia-PASEF method [18] with sample-optimized data-independent acquisition (DIA) scan parameters. To adjust DIA-PASEF parameters optimally to each sample type (HDL, plaque), data-dependent acquisition (DDA) in PASEF mode was first performed on pooled samples. The default DIA-short-gradient acquisition method was then adjusted based on the sample-specific DDA-PASEF run using the timsControl software (Bruker Daltonics). The following parameters were modified for each sample type: m/z range (405.1\u0026ndash;1255.1); mobility range (0.85\u0026ndash;1.30 1/K0); mean cycle time (1.80 s.). The ion mobility windows were optimized to best match the ion cloud density from the sample-type-specific DDA-runs. To analyze DIA-PASEF data, the raw (.d) files were processed using DIA-NN v18.0 [19] with a spectral library generated from the UniProt human proteome (UP000005640, downloaded 4.5.2022 as a FASTA file, 20378 proteins). The following settings were applied during library generation: fixed modifications: carbamidomethyl (C); variable modifications: acetyl (protein N-term), oxidation (M); enzyme: Trypsin/P; maximum missed cleavages: 1; mass accuracy: 1.5e-05 (MS2) and 1.5e-05 (MS1); fragment m/z: 100\u0026ndash;1700; peptide length: 7\u0026ndash;30; precursor m/z: 300\u0026ndash;1600; precursor charge: to 2\u0026ndash;4; protein inference: not performed. All other settings were set to default.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical analysis of the proteomics data\u003c/h3\u003e\n\u003cp\u003eThe DIA-NN Report.pg_matrix file was used as the input to further DIA data analysis. Data pre-processing was performed using an in-house R-script. Raw intensity values were log\u003csub\u003e2\u003c/sub\u003e-transformed and median-normalized. Missing values were imputed using QRILC imputation. For sample group comparison, p-values were calculated using Student\u0026rsquo;s t-test with SciPy package [20] in Python. Multiple testing correction was applied using Benjamini\u0026ndash;Hochberg method via the Statsmodels package. Volcano plots were generated using q-value threshold of 0.01 and log\u003csub\u003e2\u003c/sub\u003e fold-change thresholds of \u0026plusmn;\u0026thinsp;1.\u003c/p\u003e\n\u003ch3\u003eLipid mediator analysis\u003c/h3\u003e\n\u003cp\u003eEDTA-plasma samples (0.5 ml) were mixed with 2 ml of ice-cold methanol (LiChrosolv\u0026reg;, Merck) containing internal standards (500 pg each: d\u003csub\u003e8\u003c/sub\u003e-5-HETE, d\u003csub\u003e4\u003c/sub\u003e-Leukotriene B\u003csub\u003e4\u003c/sub\u003e, d\u003csub\u003e4\u003c/sub\u003e-PGE\u003csub\u003e2\u003c/sub\u003e, d\u003csub\u003e5\u003c/sub\u003e-LXA\u003csub\u003e4\u003c/sub\u003e, d\u003csub\u003e5\u003c/sub\u003e-RvD2; all from Cayman Chemical). Lipid mediators were then extracted following previously described principles [21]. Samples were precipitated at -20\u0026deg;C for 45 min, centrifuged at 1900xg for 10 min at 4\u0026deg;C, and supernatant was evaporated under nitrogen stream until reduced to 1 ml. Next, 9 ml of pH 3.5 MQ water was added, and samples were immediately loaded on Sep-Pak Vac 6 cc C18 cartridges (500 mg, Waters) in Waters Extraction Manifold. The solid phase extraction cartridges were equilibrated with 12 ml of methanol and 6 ml of MQ water. After sample loading, cartridges were washed with 4 ml of MQ water and 10 ml of hexane (LiChrosolv\u0026reg;, Merck). Lipid mediators were eluted with 8 ml of methyl format (Spectro Grade 98%, Fisher Scientific) and evaporated to dryness under a nitrogen stream. The dried samples were resuspended in 40 \u0026micro;l of methanol (Optima\u0026trade;, Fisher Scientific):MQ water 1:1 (vol/vol). The samples were spun at 10000xg at 4\u0026deg;C. The clear supernatant was transferred into a new insert.\u003c/p\u003e \u003cp\u003eThe whole sample was injected into 1290 Infinity II LC (Agilent technologies) system equipped with a Kinetex 2.6 \u0026micro;m C18 100 \u0026Aring; LC column (150 x 2.1 mm, Phenomenex) and coupled to a 6500\u0026thinsp;+\u0026thinsp;QTrap mass spectrometer (AB Sciex). The flow rate was set to 0.200 ml/min, and the column temperature was maintained at 50\u0026deg;C. The 33-min LC run was as follows: the mobile phase (methanol/water/acetic acid of 20:80:0.01, vol/vol/vol) was ramped to 50:50:0.01 (vol/vol/vol) over 0.75 min, next to 80:20:0.01 from 3 min to 16.5 min, maintained until 21.75 min, and then ramped to 98:2:0.01 over 0.25 min. The gradient was maintained at 98:2:0.01 from 21.9 to 30 min before returning to the initial 20:80:0.01 composition. Standards were purchased from Cayman Chemical, and the MRM transitions.\u003c/p\u003e \u003cp\u003eThe data were analyzed with Sciex OS 2.0, following previously published parameters [21] including 1) peak area\u0026thinsp;\u0026gt;\u0026thinsp;2000 counts, 2) retention time matching with an authentic reference standard, 3) minimum of 4 data points, 4) matching of at least 6 diagnostic ions to a reference standard including at least one backbone fragment from a representative subset of samples, and 5) signal-to-noise ratio\u0026thinsp;\u0026gt;\u0026thinsp;5. Peaks with signal-to-noise ratio above 5 were included in the analysis and peaks with signal-to-noise ratio between 3\u0026ndash;5 are marked in Table S2 as \u0026ldquo;Trace\u0026rdquo;.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical methods\u003c/h2\u003e \u003cp\u003eShapiro-Wilk test was used for normality testing. Normally distributed data with n\u0026thinsp;\u0026gt;\u0026thinsp;13 were analyzed by Pearson\u0026rsquo;s correlation analysis, while Spearman\u0026rsquo;s analysis was used for data sets without a normal distribution. For sample sizes n\u0026thinsp;\u0026lt;\u0026thinsp;14 correlation analysis was performed using Kendall\u0026rsquo;s Tau-P. Data were adjusted to the age and sex of patients. Immunofluorescence intensities were adjusted based on the control images in Zen 3.1 (Lite) software. One-way ANOVA with Dunnett\u0026rsquo;s test was used for multiple comparisons of unequally distributed samples. Mann-Whitney U test was used for pairwise comparisons while Student\u0026rsquo;s t-test was used for sample sets with normal distribution. Partial Spearman correlation was used to assess the relationships between variables, adjusting for age and sex using the a custom Python script. Pearson's and Kendall's correlations were calculated using SPSS version 29.0.2.0 (20).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMDA modification of HDL is associated with reduced complement regulation in patients with atherosclerosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt has been shown previously that preventing the MDA modification of HDL significantly increases plaque stability in a mouse model of atherosclerosis [22]. To better understand the role of MDA-HDL modification in humans, we analyzed MDA-HDL levels in 17 PAD patients who had undergone femoral artery endarterectomy, a surgical procedure for the removal of plaque buildup [14] (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We did not observe a correlation between MDA-HDL and HDL levels (Pearson\u0026rsquo;s \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.265, p\u0026thinsp;=\u0026thinsp;0.360; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which is consistent with previous studies showing that plasma HDL levels do not necessarily predict HDL functionality, despite their association with cardiovascular disease risk [23]. Furthermore, the levels of MDA-HDL modification did not significantly correlate with markers of complement activation (Fig. S4A), suggesting that the MDA-HDL modifications may not reflect systemic inflammation levels, but rather indicate the inflammatory status of specific locations, such as arterial walls and liver, where HDL performs its primary functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause CFH binding to MDA-modified epitopes [7] and ApoE [8] is known to protect HDL from oxidative stress and complement attack [10, 24], we next analyzed whether levels of CFH bound to HDL (CFH-HDL) correlated with MDA-HDL levels. Western blot (WB) confirmed the presence of CFH and ApoE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig. S4B) in HDLs isolated from the patients (Fig. S4B). We revealed that MDA-HDL levels negatively correlated with CFH-HDL levels measured by WB (Pearson\u0026rsquo;s \u003cem\u003er\u003c/em\u003e = -0.774, p\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and by ELISA (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e = -0.723, p\u0026thinsp;=\u0026thinsp;0.0015, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Despite the known interaction of CFH with MDA-modified epitopes, the observed negative correlation suggests that CFH binding to HDL is not dependent only on MDA modifications. Indeed, we found a positive correlation between CFH-HDL and C3b deposits on HDL (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.630, p\u0026thinsp;=\u0026thinsp;0.009, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This correlation was significant in patients with ApoE-ε4 allele (Kendall\u0026rsquo;s \u003cem\u003eτ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.511, p\u0026thinsp;=\u0026thinsp;0.04) but not with non-ε4 ApoE alleles (Kendall\u0026rsquo;s \u003cem\u003eτ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.536, p\u0026thinsp;=\u0026thinsp;0.21, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Notably, no correlation was observed between MDA-HDL and C3b deposition (Pearson\u0026rsquo;s \u003cem\u003er\u003c/em\u003e = -0.206, p\u0026thinsp;=\u0026thinsp;0.481, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting that HDL particles without MDA modification are better protected by CFH under conditions of excess complement C3b, as expected in patients with atherosclerosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCFH-mediated protection of HDL shifts HDL proteome toward cardioprotective pathways and increases cholesterol efflux.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt has been shown previously that MDA modification of HDL affects its anti-inflammatory properties by altering the HDL proteome [25]. This alteration includes the enrichment of HDL proteins involved in lipid metabolism, oxidative stress, and complement activation [26]. To assess whether HDL proteomes in our PAD patients were affected by MDA modification and whether increased CFH-HDL levels promote HDL\u0026rsquo;s anti-inflammatory properties, we used mass spectrometry to compare three samples with high CFH-HDL levels to three samples with low CFH-HDL levels. Signaling pathway analysis identified several canonical pathways directly associated with vascular health (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Among significant pathways, we found: 1) Plasma lipoprotein assembly, remodeling, and clearance, 2) Liver X receptor (LXR) activation, 3) DHCR24 (24-dehydrocholesterol reductase) signaling, and 4) Atherosclerosis signaling. LXR-mediated signaling is known to play a protective role in atherosclerosis by regulating lipid metabolism, promoting reverse cholesterol transport, and suppressing inflammation in the vessel wall. Previous studies have shown that selective LXR activation in macrophages reduced atherosclerotic lesions, improved plasma lipid profiles, and reduced cholesterol accumulation in the mouse model of atherosclerosis [27]. DHCR24 has also been previously implicated in vascular health due to its involvement in cholesterol biosynthesis, oxidative stress reduction, and endothelial protection [28].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA detailed analysis of HDL proteomic signatures revealed increased expression of proteins associated with vascular health and anti-atherogenic pathways in the high CFH-HDL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Dataset 1). Among the proteins enriched\u0026thinsp;\u0026gt;\u0026thinsp;2-fold, we identified AP2A2 (Log\u003csub\u003e2\u003c/sub\u003e fold change, Log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;5.7), a subunit of the adaptor-related protein complex 2 (AP2), which regulates macrophage cholesterol trafficking and inflammatory activity [29]. Additionally, upregulation of glutaredoxin-1 (GLRX) (log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;1.2) has been previously linked to reduced plaque burden and cardiovascular disease severity in mice [30], while RAB3D (log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;2.6) is known to be downregulated in injured carotid arteries [31]. Interestingly, two proteins significantly enriched in the high CFH-HDL group (CAMP and AP2A2) possess antimicrobial activities, supporting the long-standing hypothesis that HDL plays a role in immune defense [32, 33]. Thus, pathway analysis revealed that PAD patients with high CFH-HDL demonstrate proteome profiles associated with vascular protection, lipid metabolism, and antimicrobial activity, assuming their potential role in slowing atherosclerosis.\u003c/p\u003e \u003cp\u003eSince our analysis of signaling pathways identified several potential mechanisms involved in cholesterol trafficking, we examined whether CFH binding to HDL helps protect HDL function by enhancing cholesterol efflux. Indeed, HDL samples from patients with high CFH-HDL levels showed enhanced cholesterol efflux compared to HDL samples from patients with low CFH-HDL levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To further explore the effects of CFH-mediated complement regulation of HDL, we exposed HDL to complement attack. In the absence of CFH, complement exposure led to C3b deposition on HDL (Fig. S2B) and a significant reduction of HDL-mediated cholesterol efflux (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As expected, adding CFH to the reaction decreased C3b deposition on HDL and preserved its efflux capacity. These findings align with our mass spectrometry data, suggesting that increased CFH-HDL levels enhance HDL function.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFemoral plaque proteome reveals stability biomarkers linked to elevated CFH-HDL levels.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous results, which showed no correlation between MDA-HDL levels and plasma markers of complement activation (Fig. S4A), suggest that inflammation is localized in arterial walls near atherosclerotic plaques rather than systemically. To further investigate the relationship between circulating HDL composition and local atherosclerotic lesions in PAD patients, we used mass spectrometry to analyze proteomic profiles of femoral plaques dissected from the same group of six patients with high and low CFH-HDL levels as in HDL proteome analysis. Comparison of the plaque proteomes revealed differential expression of four proteins: HSPG2 (aka perlecan, Log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;2.2), LIMCH1 (Log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;4.5), NEGR1 (Log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;5.6), and COL18A1 (Log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026asymp;\u0026thinsp;2.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Dataset 2). According to the signaling pathway analysis, these proteins are involved in extracellular matrix organization and collagen biosynthesis/degradation, suggesting a role in plaque stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Table S3). A closer examination of the up- and downregulated proteins in each indicated pathway revealed a correlation between the protein levels and plaque stability markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Table S3). For example, perlecan and COL18A1 (Collagen, Type XVIII, Alpha 1), both indicative of stable plaques [34\u0026ndash;36], were enriched in plaques of patients with high CFH-HDL levels [37].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePerlecan is a heparan sulfate proteoglycan found in the basement membrane, where it interacts with CFH [38] and PRELP (proline/arginine-rich end leucine-rich repeat protein) [39], both of which can prevent complement attack. Since our analysis of the plaque proteomes revealed elevated perlecan levels in the high CFH-HDL group, we investigated the relevance of this finding by measuring serum perlecan levels in the PAD patients. We found no correlation between MDA-HDL and serum perlecan levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting that local perlecan concentration within plaques is more relevant than systemic perlecan levels.\u003c/p\u003e \u003cp\u003ePerlecan is also known to reflect endothelial damage and cardiovascular complications in atherosclerosis patients with chronic kidney disease (CKD) [40]. Interestingly, CFH-HDL levels showed a suggestive correlation with platelet counts (Table S4) indicating that CFH-HDL may reduce the inflammatory load and, in turn, decrease plaque instability. Serum perlecan levels correlated positively with creatinine levels (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.550, p\u0026thinsp;=\u0026thinsp;0.0016, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and negatively with glomerular filtration rates (GFR) (Pearson\u0026rsquo;s \u003cem\u003er\u003c/em\u003e = -0.547, p\u0026thinsp;=\u0026thinsp;0.003, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Since GFR and creatinine are markers of kidney function, this suggests that perlecan may serve as a possible marker for CKD in atherosclerosis patients [40]. However, serum perlecan levels did not differ between the high and low CFH-HDL groups, indicating that they reflect kidney function in atherosclerosis patients rather than plaque stability. We also observed that individuals carrying one ε4 allele in ApoE heterozygous (ApoE-ε3/ε4) had significantly higher perlecan levels than those with the ε2 or ε3 alleles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). This finding suggests that ApoE4 may increase the risk of endothelial damage, possibly due to its inability to bind CFH [10]. The role of LIMCH1 (LIM and Calponin Homology Domains 1) in atherosclerosis remains largely unexplored, but its downregulation has been observed in ruptured atherosclerotic samples from carotid arteries [41].\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh CFH-HDL levels are associated with a plaque-stabilizing sheet calcification pattern.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePlaque calcification develops through inflammation-driven mechanisms that influence atherosclerosis progression \u003csup\u003e54\u003c/sup\u003e. It varies dynamically, with nodular, sheet, and ectopic bone patterns marking disease chronicity and instability, and can be used as an independent predictor of atherosclerosis-related cardiovascular events \u003csup\u003e55\u003c/sup\u003e. Nodular calcification increases thrombotic risk via cap disruption; sheet calcification stabilizes plaques but increases their rigidity; ectopic bone reflects chronic inflammation while lowering rupture risk. Given the role of HDL in calcification of vascular cells, we examined calcification patterns \u003csup\u003e56\u003c/sup\u003e and their association with CFH-HDL and MDA-HDL levels in PAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A significant correlation was observed between CFH-HDL levels and sheet calcification (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e = -0.573, p\u0026thinsp;=\u0026thinsp;0.032) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Suggestive but not significant negative association was observed between CFH-HDL levels and nodular calcification or ectopic lamellar bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). MDA-HDL did not correlate with either nodular/sheet calcification or lamellar bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). The observed positive correlation between CFH-HDL and sheet calcification suggests a plaque-stabilizing role, potentially reducing instability and thrombotic risk in atherosclerosis. In contrast, MDA-HDL showed no significant association with sheet or nodular calcification, indicating a lesser role in plaque stability, though its correlation with bone calcification, previously linked to chronic inflammation, may reflect prolonged disease activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution of CFH-HDL, ApoE, and membrane attack complex C5b-9 in femoral plaques of PAD patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn atherosclerosis, infiltrating macrophages in the intima take up oxidized LDL, forming foam cells that contribute to plaque buildup. HDL helps remove cholesterol from these macrophages, but its function can be impaired in diseased vessels, reducing its ability to prevent foam cell formation and inflammation. Previously, we demonstrated that CFH binding to ApoE, which is associated with HDL in human plasma, protects HDL from complement attack and thereby contributes to its anti-inflammatory properties [8]. The observed negative correlation between CFH-HDL and MDA-HDL in PAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C), along with prior findings linking MDA-HDL inhibition to plaque stability [22], led us to examine the distribution of CFH-HDL, ApoE, and the membrane attack complex C5b-9 in femoral plaques. Cryosections of femoral plaques from three patients with high CFH-HDL levels and three patients with low CFH-HDL levels were stained with hematoxylin-eosin (H\u0026amp;E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig. S5), while adjacent sections were stained with antibodies against CFH, ApoE, and C5b-9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig. S3). Parallel histological and immunohistochemical analyses revealed that CFH and ApoE were primarily colocalized in plaques from high CFH-HDL patients, particularly at the periphery of fibrotic-necrotic plaque cores and in pericytic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Morphological assessment combined with DAPI nuclear counterstaining confirmed cellular association of the proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Fig. S3). Notably, C5b-9 deposition exhibited spatial correlation with CFH-HDL and ApoE in high CFH-HDL plaques, providing direct histological evidence that CFH-ApoE interaction modulates localized complement activation within atherosclerotic lesions. These findings align with established mechanisms, whereby CFH binding to HDL-associated ApoE enhances complement regulation while promoting cholesterol efflux from macrophage foam cells [24].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSynergistic effect of CFH-HDL and specialized pro-resolving mediators on vascular inflammation in atherosclerosis patients\u003c/h2\u003e \u003cp\u003ePro-inflammatory mediators contribute to ongoing inflammation in atherosclerotic plaques, promoting plaque instability and increasing the risk of cardiovascular events [11, 42]. In contrast, specialized pro-resolving mediators (SPMs), play a crucial role in resolving inflammation, stabilizing plaques and preventing rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To explore the potential synergistic interaction between SPMs and CFH-HDL in resolving inflammation in atherosclerotic plaques, we measured circulating lipid mediator levels in PAD patients using mass spectrometry, and assessed their correlation with previously determined biochemical markers and plaque calcification patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCFH-HDL levels positively correlated with plasma levels of SPM 15-epi-LXA\u003csub\u003e4\u003c/sub\u003e (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.800, p\u0026thinsp;=\u0026thinsp;0.0006, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and its precursor 15-HETE (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.574, p\u0026thinsp;=\u0026thinsp;0.025) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The levels of lipid mediators and their pathway markers did not correlate with the MDA-HDL levels, although a trend to an inverse association was observed for the 15-HETE pathway mediators (sum of lipoxins (LX) and 15-HETE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Further analysis showed that sheet calcification, exhibited a positive correlation with 15-HETE (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.596, p\u0026thinsp;=\u0026thinsp;0.025), and 17-HDHA (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.661, p\u0026thinsp;=\u0026thinsp;0.010), (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G). Moreover, the potent anaphylatoxin, C5a, correlated positively with PGD2 (Spearman\u0026rsquo;s \u003cem\u003eρ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.741, p\u0026thinsp;=\u0026thinsp;0.0016) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). While several of these lipid mediators have protective effects in atherosclerosis [43] or are metabolites of SPM pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), PGD2 has a dual role as a pro-resolving and pro-inflammatory mediator. Thus, these data suggest that the general increase in pro-resolving lipid mediators and the presence of HDL-associated CFH refers to a more responsive regulation of inflammation.\u003c/p\u003e \u003cp\u003eThese findings suggest that CFH-HDL levels may influence both the distribution patterns and extent of atherosclerosis in PAD patients. However, given the small sample size, these observations warrant further investigation with larger cohorts to establish their significance. To sum up, our data suggest a synergy between CFH-HDL and SPMs as anti-inflammatory and anti-atherogenic molecules, which may serve as a potential target for inhibiting plaque instability. Along with other identified biomarkers, these factors could potentially be used for diagnosing individuals with high-risk, rupture-prone atherosclerotic plaques.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAtherosclerotic plaque structure significantly affects its susceptibility to rupture, and therefore, understanding the key factors determining the stability of the plaque is essential. Our analysis of the oxidative modification, the protein profile of circulating HDL, and plasma metabololipidome, shows that the circulating molecular profiles markedly mirror plaque histomorphology, and their inflammatory microenvironment. HDL has a targeted function on the artery wall and the potential to prevent the development of atherosclerosis. We analyzed the presence of MDA adducts and CFH levels in circulating HDL of PAD patients and discovered immune molecules and structural components of plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) strongly implying human plaque instability, as evidenced by the plaque proteome, histological features, and metabololipidome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe observations that high levels of plasma lipid mediators and their pathway markers were associated with increased CFH-HDL levels, plasma complement activation markers C3a and C5a, and sheet calcification in plaques are novel. Apparently, the complement cues of inflammation and counteracting responses effectively enhance the SPM production in certain patients to attenuate the inflammation [43]. Since the levels of the markers of SPM pathways 15-HETE and 17-HDHA showed an inverse trend with MDA-HDL, the SPMs may also help mitigate oxidative stress [45]. These observations highlight the pathophysiological role of active lipid mediator biosynthesis in promoting plaque stability through their pro-resolving functions.\u003c/p\u003e \u003cp\u003eThe major histological markers for plaque instability include atherosclerotic lesion collagen content, fibrous cap thickness, and nodular calcification. Previous studies indicate that calcified nodules and superficial calcifications correlate with plaque vulnerability, whereas substantial and widespread calcifications promote plaque stabilization [46]. Transcriptomic studies show that highly calcified, stable plaques are associated with the upregulation of smooth muscle-related pathways and downregulation of inflammation. Previous proteomic and transcriptomic analyses of arterial plaques in coronary artery disease and carotid artery disease have identified several factors implying plaque vulnerability including extracellular matrix proteins, genetic markers, and inflammatory macrophages [35] [47]. Although some of these proteins/pathways have been implicated before, our observation that these immune molecules and structural components of plaques significantly associate with patient CFH-HDL levels is novel and thereby highlighting important roles for complement regulation in atherosclerosis. Furthermore, we provide mechanistic rationale (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D) for the anti-atherogenic and anti-inflammatory functions of HDL-associated CFH.\u003c/p\u003e \u003cp\u003eIn atherosclerosis, MDA-HDL epitopes are important mediators for inflammation and potential targets for therapeutic interventions. On atherosclerotic plaques, however, CFH binds to MDA-LDL and thereby reduces the pro-inflammatory effects of MDA in a murine model [7]. Interestingly, we observed that in ApoE4-carriers increased C3b depositions on HDL resulted in a corresponding increase in CFH levels. These increased CFH-HDL levels correlated positively with the pro-resolving 15-epi-LXA\u003csub\u003e4\u003c/sub\u003e and its pathway marker 15-HETE. These mediators promote resolution by blocking neutrophil chemotaxis and transepithelial migration, decreasing the NF-κB signaling and production of pro-inflammatory cytokines, and, importantly, increasing efferocytosis, the clearance of apoptotic cells [48]. These findings suggest that CFH-HDL may help protect against the disease by counteracting ongoing inflammation. Furthermore, CFH has the capacity to facilitate clearance of cellular debris which is characteristic in this disease. The crucial role of CFH in the non-inflammatory clearance is exemplified by the Y402H polymorphism in CFH, where impaired complement regulation leads to metabolic debris and drusen accumulation in age-related macular degeneration (AMD) [10]. Moreover, CFH\u0026rsquo;s inability to bind the Alzheimer\u0026rsquo;s disease-associated ApoE4 increases Aβ oligomerization, promoting plaque formation and neuroinflammation in Alzheimer\u0026rsquo;s disease [10].\u003c/p\u003e \u003cp\u003eThe importance of CFH in regulating complement in various tissues can be attributed to its soluble nature, allowing it to reach areas where complement activation is exacerbated, such as atherosclerotic plaques. Here we provide further evidence of its role in protection against complement in atherosclerotic plaques \u003cem\u003eex vivo\u003c/em\u003e, as evidenced by the immunofluorescence microscopy demonstrating colocalization between ApoE, CFH, and C5b-9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, the suggestive negative correlation between patient CFH-HDL levels and platelet counts indicates that reduced CFH levels may promote chronic inflammation and platelet activation, both of which are known to increase the risk of cardiovascular events in atherosclerosis patients [49]. Furthermore, the protective role of CFH in atherosclerosis is supported by evidence that CFH V62I polymorphism correlates with the serum levels of matrix metalloproteinase 8 (MMP-8), which is a pro-inflammatory enzyme linked to cardiovascular diseases [50].\u003c/p\u003e \u003cp\u003eThe strengths of our study include a novel approach to identifying a comprehensive set of immune molecules and structural components of plaques through high-throughput mass spectrometry methods covering the proteome and lipidome of patient HDLs, serum and atherosclerotic plaques, and metabololipidome of plasma. Our study has limitations. First, ultracentrifugation reduces the detection of low-affinity proteins that could be crucial to HDL\u0026rsquo;s function. In addition, we did not differentiate between HDL subpopulations that may differ in their anti-atherogenic functions. Second, our study population of 17 patients is small, and verification of these results requires studies with larger patient cohorts. Furthermore, the patients studied had advanced atherosclerotic disease requiring invasive treatment. Additional work is required to determine the pathways by which complement regulation and pro-resolving responses are linked.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eBy using advanced proteomic and metabololipidomic profiling of patient HDL, plasma, and femoral artery plaques, this study provides molecular insights into the inflammatory mechanisms underlying plaque rupture. Importantly, CFH-HDL concentrations correlated positively with both pro-resolving lipid-mediator pathways and increased extracellular matrix proteins, while inverse patterns were linked to plaque instability. Furthermore, we demonstrated that CFH on HDL increased the capacity of HDL to promote cholesterol efflux, providing direct mechanistic evidence for CFH-HDL as an anti-atherogenic molecule. These findings suggest that complement regulation by CFH and the pro-resolving mechanisms of SPMs work synergistically to promote plaque stability and counteract extracellular matrix breakdown. Our results underscore the potential of circulating biomarkers to reflect plaque pathology, offering clinically relevant insights into mechanisms of plaque stability and pointing to new therapeutic strategies for managing atherosclerotic disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplement factor H\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-density lipoprotein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperipheral arterial disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDL-C\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHDL-cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow-density lipoprotein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eApoE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eapolipoprotein E\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003especialized pro-resolving mediators\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,3',5,5'-Tetramethyl- - benzidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEthylenediaminetetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSPG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eheparan sulpfate proteoglycan\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhorbol 12-myristate 13-acetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and Eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrifluoroacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDIA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edata-independent acquisition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edata-dependent acquisition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePASEF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparallel accumulation\u0026ndash;serial fragmentation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQRLIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantile regression approach for left-censored missing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHETE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHydroxyeicosatetraenoic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstaglandin E2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLXA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eA lipoxin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRvD2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eResolvin D2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMilli Q\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLXR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiver X receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHCR24\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e24-dehydrocholesterol reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAP2A2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAP-2 complex subunit alpha-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAP2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadaptor-related protein complex 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGLRX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglutaredoxin-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRAB3D\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRas-related protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCyclic adenosine monophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLIMCH1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLIM and calponin homology domains-containing protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNEGR1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeuronal growth regulator 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOL18A1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecollagen type XVIII alpha 1 chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRELP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eproline/arginine-rich end leucine-rich repeat protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echronic kidney disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglomerular filtration rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e15-epi-LXA\u003csub\u003e4\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e15-epi-lipoxin A4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDocosahexaenoic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emagnetic resonance angiogram\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP-8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ematrix metalloproteinase 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate and consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;study protocol was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa (ASO‐project 78/13/03/00/2014), and all procedures were conducted in accordance with applicable guidelines and regulations.Informed consent for publication has been obtained from participants or their legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: KH, EN, SS, PU\u003c/p\u003e\n\u003cp\u003eMethodology: EN, SS, PU, IL, ML, LC, ER, EV, AT, SK, CM, MH\u003c/p\u003e\n\u003cp\u003eInvestigation: KH, EN, SS, PU, IL, ML, LC, ER, EV, AT, SK, CM, MM, MH, RK, JS\u003c/p\u003e\n\u003cp\u003eVisualization: KH, EN, SS, PU, IL, ML, CM, MH, JS\u003c/p\u003e\n\u003cp\u003eFunding acquisition: KH\u003c/p\u003e\n\u003cp\u003eProject administration: KH\u003c/p\u003e\n\u003cp\u003eSupervision: KH, MV, JS, RK\u003c/p\u003e\n\u003cp\u003eWriting – original draft: KH, EN, SS, PU\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing: KH, EN, SS, PU, IL, ML, LC, ER, EV, AT, SK, CM, MM, SM, MH, MV, JS, RK\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Finnish foundation for cardiovascular research (190044 and 230023)\u003c/p\u003e\n\u003cp\u003eAcademy of Finland (1331108)\u003c/p\u003e\n\u003cp\u003eJane and Aatos Erkko foundation (190002)\u003c/p\u003e\n\u003cp\u003eIda Montin foundation\u0026nbsp;(2021)\u003c/p\u003e\n\u003cp\u003eBiomedicum Helsinki foundation (20220141).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Matti Jauhiainen (Minerva Institute, Helsinki, Finland) for reviewing the manuscript and providing the rabbit anti-apoE antibody.\u003c/p\u003e\n\u003cp\u003eDr. Anastasia Ludwig (Neuroscience Center, HiLIFE, Helsinki, Finland) for providing the Python script for statistical analyses.\u003c/p\u003e\n\u003cp\u003eLauri Snellman, Vellamo Tuomainen and Juuso Vedenjuoksu for their excellent assistance in the laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInfrastructures Bioimaging unit (BIU), University of Helsinki, Finland.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e Data are available in the main text or the supplementary materials. Proteomic data files will be uploaded in MassIVe with ID numbers.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAday AW, Everett BM: \u003cstrong\u003eDyslipidemia Profiles in Patients with Peripheral Artery Disease\u003c/strong\u003e. \u003cem\u003eCurr Cardiol Rep \u003c/em\u003e2019, \u003cstrong\u003e21\u003c/strong\u003e(6):42.\u003c/li\u003e\n\u003cli\u003eSyed S, Nissila E, Ruhanen H, Fudo S, Gaytan MO, Sihvo SP, Lorey MB, Metso J, Oorni K, King SJ\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eStreptococcus pneumoniae pneumolysin and neuraminidase A convert high-density lipoproteins into pro-atherogenic particles\u003c/strong\u003e. \u003cem\u003eiScience \u003c/em\u003e2021, \u003cstrong\u003e24\u003c/strong\u003e(6):102535.\u003c/li\u003e\n\u003cli\u003eKhera AV, Cuchel M, de la Llera-Moya M, Rodrigues A, Burke MF, Jafri K, French BC, Phillips JA, Mucksavage ML, Wilensky RL\u003cem\u003e et al\u003c/em\u003e: 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Cardiovascular Diseases: A Genome-Wide Analysis\u003c/strong\u003e. \u003cem\u003eCirc Cardiovasc Genet \u003c/em\u003e2017, \u003cstrong\u003e10\u003c/strong\u003e(6).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Complement, inflammation, lipidomics, proteomics","lastPublishedDoi":"10.21203/rs.3.rs-6828685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6828685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInflammation is a key driver of plaque rupture and adverse cardiovascular events in atherosclerosis patients. However, accurate identification of high-risk individuals in preventative cardiology has remained elusive.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study we analyzed the biochemical characteristics of high-density lipoproteins (HDL), plasma metabololipidomics and atherosclerotic plaques obtained from peripheral artery disease patients. This included the measurements of circulating complement activation markers, complement factor H-associated HDL, malondialdehyde modified HDL, plasma lipid mediators, as well as analyzing the HDL and plaque proteome. In addition, arterial plaques were subjected to histological and immunofluorescence staining.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe identified novel pro- and anti-inflammatory molecules and structural components of arterial plaques indicative of plaque stability. An increase in HDL-associated complement regulatory protein factor H correlated with higher levels of specialized pro-resolving lipid mediators, changes in plaque calcification, and specific extracellular matrix proteins. The presence of factor H in HDL was associated with increased cholesterol efflux supporting its role in HDL\u0026rsquo;s antiatherogenic effects.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings indicate that complement regulation and pro-resolving responses work synergistically to exert anti-inflammatory effects, which are essential for maintaining extracellular matrix integrity. These findings may assist in early detection of high-risk cardiovascular disease and optimize therapeutic strategies to prevent adverse events.\u003c/p\u003e","manuscriptTitle":"Complement Factor H and Pro-Resolving Mediators Synergistically Enhance Plaque Stability in Peripheral Arterial Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 06:17:17","doi":"10.21203/rs.3.rs-6828685/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ee5c8615-1ebf-4031-904b-f4786e70f575","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-07T14:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-25 06:17:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6828685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6828685","identity":"rs-6828685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}