International Journal of Molecular Sciences Article Alternative C3 Complement System: Lipids and Atherosclerosis Maisa Garcia-Arguinzonis 1 , Elisa Diaz-Riera 1 , Esther Peña 1,2 , Rafael Escate 1,2 , Oriol Juan-Babot 1 , Pedro Mata 3 , Lina Badimon 1,2,4,† and Teresa Padro 1,2, *,† 1 Cardiovascular Program-ICCC, Research Institute-Hospital Santa Creu i Sant Pau, IIB-Sant Pau, 08025 Barcelona, Spain; mgarciaar@santpau.cat (M.G.-A.); ediazr@santpau.cat (E.D.-R.); epena@santpau.cat (E.P.); rescate@santpau.cat (R.E.); ojuan@santpau.cat (O.J.-B.); lbadimon@santpau.cat (L.B.) 2 Centro de Investigación Biomédica en Red Cardiovascular (CIBERCV), Instituto de Salud Carlos III, 28029 Madrid, Spain 3 Fundación Hipercolesterolemia Familiar, 28010 Madrid, Spain; pmata@colesterolfamiliar.org 4 Cardiovascular Research Chair, UAB, 08025 Barcelona, Spain * Correspondence: tpadro@santpau.cat; Tel.: +34-935-565-886; Fax: +34-935-565-559 † Both authors contributed equally. Abstract: Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a pheno- type that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on  the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth  muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular Citation: Garcia-Arguinzonis, M.; matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, Diaz-Riera, E.; Peña, E.; Escate, R.; with a significantly different proteomic signature in AT segments. Thus, C3 products were more Juan-Babot, O.; Mata, P.; Badimon, L.; abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels Padro, T. Alternative C3 Complement were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed System: Lipids and Atherosclerosis. Int. J. Mol. Sci. 2021, 22, 5122. tomographic angiography. However, no correlation was identified between circulating C3 levels and https://doi.org/10.3390/ijms22105122 the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and Academic Editors: Maria I. the integrin αM β2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated Dorobantu and Maya Simionescu in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and Received: 17 April 2021 iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of Accepted: 6 May 2021 VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin Published: 12 May 2021 stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in Publisher’s Note: MDPI stays neutral vascular remodeling and in the progression of advanced human atherosclerotic lesions. with regard to jurisdictional claims in published maps and institutional affil- Keywords: atherosclerosis; cardiovascular disease; complement system; proteomics; mass spectrom- iations. etry Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Familial hypercholesterolemia (FH), an autosomal-dominant disorder mainly caused distributed under the terms and by the loss-of-function mutations in the low-density lipoprotein (LDL) receptor, is asso- conditions of the Creative Commons ciated with an increased risk of atherosclerosis and ultimately premature cardiovascular Attribution (CC BY) license (https:// event presentation, resulting in lifelong exposure to high-LDL cholesterol levels [1–5]. creativecommons.org/licenses/by/ Increasing evidence suggests that FH patients recurrently present an inflammatory phe- 4.0/). notype that is maintained despite treatment with lipid lowering therapies according to Int. J. Mol. Sci. 2021, 22, 5122. https://doi.org/10.3390/ijms22105122 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5122 2 of 17 guidelines [6–9]. We and others have demonstrated that adult FH patients have higher lev- els of extracellular microvesicles originating from inflammatory cells in plasma [6] as well as circulating mononuclear cells and monocyte-derived macrophages with inflammatory phenotypes [7,10]. The complement system is an important component of the innate immunity and plays a key role in the regulation of inflammation. Particularly relevant is the activation of the alternative C3 system, in which the different pathways of the complement system converge, leading to the formation of active C3 proteolytic products, C5 convertases and eventually the activation of the terminal complement proteins C5 to C9 and the formation of the membrane attack complex (MAC) [11,12]. The C3 system is tightly regulated by a cascade of components, including activators (Factor B), inhibitors (Factor H) and cell surface proteins acting as receptors (CR1, C3aR and αM β2 integrin). Atherosclerosis is widely recognized as a lipid-induced chronic inflammatory disease of the arterial wall with the activation of resident cells and recruitment of circulating leukocytes [13]. The complement system has been repeatedly associated with vascular remodeling [14] and atherosclerosis [15]. Results from experimental animal models and human samples suggest that complement activation may exert dual atheroprotective and proatherogenic effects mainly associated with the initial and terminal stages of the complement cascade, respectively [16,17]. However, the impact of the C3 complement system in atherogenesis is not fully understood. C3 complement products and their cell receptors have been detected by immunohisto- chemistry in areas with atherosclerotic lesions of different severity in human arteries [18,19], which has led to the hypothesis that local activation of the alternative-complement system is involved in atherosclerotic plaque progression and complication [15]. In contrast, prior studies in mice models of atherosclerosis (Ldlr-/- or ApoE-/- Ldlr-/- background) and knock-out C3 expression (C3-/-) evidenced that atherosclerotic lesions developed in the absence of C3 have a lower content of vascular smooth muscle cells (VSMCs) and collagen, hallmark of vulnerable plaques, and are of a larger size than those plaques developed in animals with a sufficient content of C3 [20,21]. A potential effect of C3 on the proliferation of VSMCs during atherogenesis was suggested by a recent study, in ApoE-/- mice fed a high-fat Western diet, in which dedifferentiated clonally expanding vascular SMC showed an up-regulated C3 expression [22] and also by prior results linking C3a with the increasing proliferation of mouse VSMCs [23]. Results from two recent studies evidenced a noticeable increase in arterial wall inflam- mation, assessed by fluorodeoxyglucose positron emission tomography imaging, in FH patients with high LDL levels with healthy controls [24,25]. The up-regulation of compo- nents of the complement cascade, including C3-derived products, have been reported in two studies in FH patients with no clinical evidence of coronary artery disease [23,26]. To date, however, we do not know whether circulating C3 levels relate to the intensity and profile of atherosclerotic plaque burden. Moreover, little is known regarding the interplay among LDL, C3 complement products and VSMCs, although VSMCs are the key cellular components in the development and complication of atherosclerotic lesions. Therefore, the present study was conducted to investigate the relationship between circulating C3 complement, lipids and atherosclerotic plaque burden in FH patients with subclinical atherosclerosis. In addition, using a mass spectrometry-based proteomic ap- proach, combined with transcriptomic analysis and in vitro functional assays, we analyzed the pattern of the C3 complement components in the extracellular matrix (ECM) of hu- man atherosclerotic plaques and investigated C3 complement expression and effects on migration kinetics of lipid-loaded VSMCs. 2. Results 2.1. Characteristics of the FH Patient Population FH patients from the SAFEHEART cohort were included (N = 49; 31 men and 18 women). The mean age was 44.7 ± 10.5 years (men: 45.4 ± 11.4 years; women Int. J. Mol. Sci. 2021, 22, 5122 3 of 17 43.7 ± 9.2 years). The baseline demographic and clinical characteristics of the studied FH-population are shown in Table 1. All subjects were on lipid-lowering treatment (LLT) and treated with statins as per the guidelines for > 1 year (mean treated years before inclusion in the study were 14.9 ± 6.7 years). The mean LDL-cholesterol (LDL-C) in the FH group was 136.3 ± 36.0 mg/dL. Table 1. Demographic, biochemical and clinical variables: Familial hypercholesterolemia and healthy subject groups. Familial Hyperchlesterolemia Healthy Subjects n = 49 n = 28 Demographic Characteristics; mean ± SD Female/male, n 18/31 16/12 Age, years 38.6 ± 11.3 24.5 ± 4.6 Risk Factors; n (%) Smokers 14 (29) 12 (43) Hypertension 1 (2) 0 (0) Diabetes mellitus 0 (0) 0 (0) Dyslipidaemia 48 (98) 0 (0) Biochemical Data, Mean ± SD Total cholesterol, mg/dL 282 ± 72 170 ± 20 Triglycerides, mg/dL 104 ± 67 77 ± 35 HDL cholesterol, mg/dL 47 ± 11 56 ± 15 LDL cholesterol, mg/dL 221 ± 78 99 ± 15 Apo AI, mg/dL 135 ± 20 139 ± 29 Apo B, mg/dL 134 ± 41 61 ± 10 Lipoprotein(a), mg/dL 42 ± 35 18 ± 21 Glucose, mg/dL 89 ± 9 78 ± 9 C-reactive protein 1.86 ± 2.6 0.73 ± 0.2 Subclinical Atherosclerotic Disease; (%) Plaque burden, % 23.5 ± 6.3 - Calcium burden, % 2.2 ± 2.5 - Non-calcium burden 21.3 ± 5.3 - BAckground Medication; n (%) Angiotensin-converting-enzyme inhibitors 0 (0) 0 (0) Angiotensin II receptor blockers 1 (2) 0 (0) Beta-blockers 0 (0) 0 (0) Diuretics 2 (4) 0 (0) Statins * 39 (80) 0 (0) SD: standard deviation * Includes: rosuvastatin, ezetimibe, atorvastatin, simvastatin, lovastatin, pravastatin, Fluvastatin, pitavastatin, resins, and fibrates. Healthy subject population was used to establish the C3 range in a healthy group. None of the FH patients had a clinical history of cardiovascular disease (CVD). Less than 5% of patients with FH presented hypertension or Type-2 diabetes. Eleven FH patients (22%) were active tobacco smokers. The mean value for 5- and 10-year CVD risk in the FH patients, according the SAFEHEART-risk score (SAFEHEART-RS), was 1.00 ± 0.76 % and 2.13 ± 1.6 %, respectively. All FH subjects included in the study presented subclinical atherosclerosis, assessed by computed tomographic angiography (CTA) and quantified by SAPC software [27]. The mean value for the total plaque burden was 23.5 ± 6.3% and, specifically, the median calcified plaque burden was 2.2 ± 2.5%, and that of non-calcified-plaque burden was 21.3 ± 5.3%. FH patients with plaque burden above the median values (high plaque burden) had a significantly higher estimated cardiovascular risk based on SAFEHEART- RS, both at 5 (0.77 ± 0.76% vs. 1.24 ± 0.15%; p = 0.03) and 10 years (1.65 ± 1.59% vs. 2.60 ± 0.15%; p = 0.03). 2.2. C3 Complement in Patients with Hypercholesterolemia and Subclinical Atherosclerosis Circulating levels of C3 complement were significantly elevated (p < 0.001) in subjects with a genetic diagnosis of FH and subclinical coronary atherosclerosis, when compared to the plasma levels of C3 in young healthy subjects at low atherosclerotic risk (subjects without CV risk factors and age between 18 and 35 years) (Figure 1A). Plasma C3 comple- ment levels were significantly correlated with LDL-C levels (Spearman correlation: Rho value = 0.412, p < 0.001), ApoB levels (Spearman correlation: Rho = 0.562, p < 0.001) and 2.2. C3 Complement in Patients with Hypercholesterolemia and Subclinical Atherosclerosis Circulating levels of C3 complement were significantly elevated (p < 0.001) in subjects with a genetic diagnosis of FH and subclinical coronary atherosclerosis, when compared to the plasma levels of C3 in young healthy subjects at low atherosclerotic risk (subjects Int. J. Mol. Sci. 2021, 22, 5122 4 of 17 without CV risk factors and age between 18 and 35 years) (Figure 1A). Plasma C3 comple- ment levels were significantly correlated with LDL-C levels (Spearman correlation: Rho value = 0.412, p < 0.001), ApoB levels (Spearman correlation: Rho = 0.562, p < 0.001) and Lp(a) (Spearman correlation: Lp(a) (Spearman correlation: Rho Rho == 0.244, 0.244, pp == 0.034) 0.034) when when the the whole whole study study population was population was considered (healthy subjects and FH patients) (Figure 1B). No significant correlation considered (healthy subjects and FH patients) (Figure 1B). No significant correlation was was found with other other lipid lipid variables, variables, including includingtriglycerides triglycerides(Spearman (Spearmancorrelation: correlation:=0.021, =0.021, p =p = 0.856) 0.856)and andHDL-cholesterol HDL-cholesterol(Spearman (Spearman correlation: correlation: Rho Rho = −0.144, −0.144, pp == 0.213). 0.213). Levels of circulating C3 in FH patients did not significantly vary in relation to the severity severity of total- plaque burden (Figure 1C). Circulating levels of C-reactive protein (CRP) in FH patients patients were below 1mg/L 1mg/L in all subjects (median [IQR] mg/L: 0.035[0.020-0.258]). in all subjects (median [IQR] mg/L: 0.035[0.020–0.258]). Circulating 1.Circulating Figure 1. C3 C3 complement complement in patients in patients with hypercholesterolemia with hypercholesterolemia and subclinical and subclinical athero- sclerosis. (A) Plasma atherosclerosis. circulating (A) Plasma C3 complement circulating (µg/mL) (µg/mL) C3 complement in subjectsinwith genetic subjects withdiagnosis genetic of FH diag- and nosissubclinical of FH and atherosclerosis (n = 49) compared subclinical atherosclerosis to levels intoalevels (n = 49) compared young inhealthy a youngpopulation (n = 28). healthy population (B)=Correlation (n between between 28). (B) Correlation plasma C3 levelsC3 plasma and LDL-C, levels andApoB LDL-C, and Lp(a) ApoB levels and in levels Lp(a) the study popula- in the study tion (FH patients with subclinical atherosclerosis and healthy population). (C) Circulating C3 com- population (FH patients with subclinical atherosclerosis and healthy population). (C) Circulating plement by plaque burden tertiles in FH patients. Dashed box (C3-NR) indicates the normal range C3 complement by plaque burden tertiles in FH patients. Dashed box (C3-NR) indicates the normal range of C3 levels in a healthy population (n = 28). Results are shown as median ± SE. p < 0.05 was considered statistically significant (Mann–Whitney and Kruskal–Wallis tests). 2.3. C3 Alternative System Components in Human Advanced Atherosclerotic Lesions Human atherosclerotic aortas were obtained from the Eulalia Study Biobank (ICCC) [28]. The extracellular matrix (ECM) of human aortas was enriched in active com- ponents of the C3 system with a significantly different proteomic signature in atheroscle- rotic areas when compared to lesion-free segments. Specifically, by two-dimensional electrophoresis (2DE) and MS/MS (MALDI-ToF/ToF), the complement-protein C3 was consistently identified as two independent spots (s1 and s2) in the ECM of atherosclerotic lesions (Figure 2A and Figure S1), whereas only weaker or non-consistent signals for s1 and s2 were identified in the ECM of aortic segments without macroscopic evidence of atherosclerotic lesions. When analyzed by Western blot (Figure 2B), protein extracts from aortic ECM showed three different C3-positive bands corresponding, based on their molec- ular size, to the full-length molecule (185 kDa), the C3 α-chain (113 kDa, obtained after proteolytic loss of a four-arginine peptide) and the final proteolytic product C3c-fragment tified as two independent spots (s1 and s2) in the ECM of atherosclerotic lesions (Figures 2A and S1), whereas only weaker or non-consistent signals for s1 and s2 were identified in the ECM of aortic segments without macroscopic evidence of atherosclerotic lesions. When analyzed by Western blot (Figure 2B), protein extracts from aortic ECM showed Int. J. Mol. Sci. 2021, 22, 5122 three different C3-positive bands corresponding, based on their molecular size, to the5 full- of 17 length molecule (185 kDa), the C3 α-chain (113 kDa, obtained after proteolytic loss of a four-arginine peptide) and the final proteolytic product C3c-fragment (39.5 kDa, α-chain). ECM extracts from atherosclerotic (AT) segments had >3-fold higher intensity in the C3- (39.5 kDa, positive α-chain). bands ECM extracts than protein extractsfrom fromatherosclerotic non-lesion (nL)(AT) segments segments (AThad vs. >3-fold higher nL: p < 0.05 for intensity in the C3-positive bands than intensity differences for each protein band).protein extracts from non-lesion (nL) segments (AT vs. nL: p < 0.05 for intensity differences for each protein band). Figure 2. C3 complement components in human atherosclerotic lesions. (A) Representative 2D-gel Figure 2. C3 complement components in human atherosclerotic lesions. (A) Representative 2D-gel images for protein extracts from normal and atherosclerotic ECM of human aortas (n = 3 independ- images for protein extracts from normal and atherosclerotic ECM of human aortas (n = 3 independent ent with nL and AT segments). Arrow heads indicate position of spots identified as C3 by MALDI- with nL and ToF/ToF AT segments). (Mascot Arrow heads indicate score = 82). Tropomyosin-1 (TMP1)position of spots identified and Haptoglobin (HPT) are as C3 by MALDI- indicated as land- ToF/ToF (Mascot score = 82). Tropomyosin-1 (TMP1) and Haptoglobin (HPT) are indicated marks. (B,C) Western Blot analysis for C3 and C3 receptors in total extracts from non-lesion as (nL) landmarks. (B,C) Western Blot analysis for C3 and C3 receptors in total extracts from non-lesion (nL) and atherosclerotic (AT) segments of human aortas (n = 4 independent arteries with nL and AT segments). (B) denotes C3 activation products, and C refers to receptors C3aR and αM β2 for C3a and iC3b/C3b (C3 activation products). Band relative intensity was normalized against total protein, visualized with Ponceau Red staining and expressed as mean ± SEM. The antibody against C3 recognizes complete full-length C3 (C3), C3 α-chain (C3 α-chain) and C3 α-chain-fragment 2 from C3c (C3c), product of degradation of iC3b. p < 0.05 was considered statistically significant (Mann–Whitney test). Moreover, C3-activated fragment receptors were consistently detected in the cell– protein fraction (SDS fraction) of aortic extracts, both from control and atherosclerotic areas (Figure 2C). Western blot analysis showed significantly higher levels of the αM and β2 subunits (CD11b and CD18, respectively) of the integrin αM β2 (iC3b/C3b receptor) in atherosclerotic segments, whereas the expression level of the anaphylatoxin C3a receptor (C3aR) did not significantly differ between non-lesion and atherosclerotic aortic segments. (Figure 2C). Moreover, C3-activated fragment receptors were consistently detected in the cell– protein fraction (SDS fraction) of aortic extracts, both from control and atherosclerotic ar- eas (Figure 2C). Western blot analysis showed significantly higher levels of the αM and β2 subunits (CD11b and CD18, respectively) of the integrin αMβ2 (iC3b/C3b receptor) in Int. J. Mol. Sci. 2021, 22, 5122 atherosclerotic segments, whereas the expression level of the anaphylatoxin C3a receptor 6 of 17 (C3aR) did not significantly differ between non-lesion and atherosclerotic aortic segments. (Figure 2C). 2DE-MS/MS analysis of human aortic ECM also evidenced the presence of C3 system 2DE-MS/MS regulatory components analysis (Figureof3A), human aortic the including ECM also evidenced complement factorthe presence H (CFH) andofCFH- C3 sys- tem regulatory components (Figure 3A), including the complement related proteins CFHR1 and CFHR5. CFHR1 and CFHR5 were identified as two inde- factor H (CFH) and CFH-related proteins CFHR1 and CFHR5. CFHR1 and CFHR5 were pendent clusters of 7 and 4 spots (MW of 37–40 and 55 kDa), respectively. CFHR1 and identified as two independent CFHR5 clusters clusters showedof 7 and 2.5- and43.8-fold spots (MW of 37–40 (average and 55spots) of cluster kDa),higher respectively. CFHR1 labeling signalsand CFHR5 clusters showed 2.5- and 3.8-fold (average of cluster spots) higher in the ECM samples from atherosclerotic segments than those from normal aortic tissue labeling signals in the (Table 2) ECM and asamples differentfrom spot atherosclerotic segments pattern distribution than those in normal from normal aortic and atherosclerotic tissue segments of the aortic vessel wall (see Figure 3A). In addition, the complement component C5 (de- of (Table 2) and a different spot pattern distribution in normal and atherosclerotic segments the aortic vessel wall (see Figure 3A). In addition, the complement component C5 (detected tected as α-chain) and its proteolytic products were consistently found in the aortic vessel as α-chain) and its proteolytic products were consistently found in the aortic vessel wall, wall, regardless of the presence of atherosclerotic lesions (Figure 3C). It is worth noting regardless of the presence of atherosclerotic lesions (Figure 3C). It is worth noting that that the relative abundance of C5 fragmentation products (C5α1 and C5α1-I) was lower the relative abundance of C5 fragmentation products (C5α1 and C5α1-I) was lower in in atherosclerotic than in apparently normal segments. atherosclerotic than in apparently normal segments. Figure Figure 3. C3 C3 complement 3. complement regulatory regulatory components components in human in human atherosclerotic atherosclerotic lesions.lesions. (A) Repre- (A) Representa- tivesentative 2D-gel(n 2D-gel images images (n = 3 independent = 3 independent with nL- with nL-segments) and AT and AT segments) of the proteomic of the proteomic pattern pattern corre- sponding to CFH, to corresponding CFHR1 CFH, (s1 to s8)(s1 CFHR1 andtoCFHR5 s8) and(s9 to s11). CFHR5 (s9Arrows to s11).indicate Arrows spots with indicate significantly spots with signif- increased icantly expression in AT-lesion increased expression in samples AT-lesion(See Table (See samples 2). (B) Scheme Table of Scheme 2). (B) the proteolytic C3 cascadeC3 of the proteolytic indicating cascadesteps regulated indicating stepsby CFHRs. (C) regulated Western(C) by CFHRs. blotWestern analysisblot of C5α chainofproteolysis analysis C5α chain products proteolysis in total extracts from non-lesion (nL) and atherosclerotic (AT) segments of human products in total extracts from non-lesion (nL) and atherosclerotic (AT) segments of human aortas (n aortas =4 (n = 4 independent arteries with nL and AT segments). Bars refer to arbitrary units of volume intensity in the Western blot bands (Mean ± SEM). Significance (p < 0.05, Mann–Whitney test) is indicated. Table 2. C3 complement-system proteins identified on advanced atherosclerotic-lesion human aortas. UniProt- Gene- Seq/Int MW Fold- Fraction Protein MS-Score * pI-Value Code Code Cov. (%) * (kDa) Change sb-ECM Complement Factor H P08603 CFH 102 9.9/78.4 143.7 6.20 ≈(1.2) Complement Factor Sb-ECM Q03591 CFHR1 120 28.2/95.3 38.8 8.70 ↑(2.4) H-related protein 1 Complement Factor Sb-ECM Q9BXR6 CFHR5 28 – 66.4 7.00 ↑(3.9) H-related protein 5 ** Lb-ECM Complement C3 P01024 C3 83 9.4/76.7 188.6 6.00 ↑(2.9) Proteins were identified by peptide mass fingerprint and confirmed by MS/MS by MALDI ToF/ToF. * Mascot Score, sequence and intensity coverage are expressed as representative values. ** Only identified by MS/MS. Complement Factor H- Sb-ECM Q9BXR6 CFHR5 28 -- 66.4 7.00 ↑ (3.9) related protein 5 ** Lb-ECM Complement C3 P01024 C3 83 9.4/76.7 188.6 6.00 ↑ (2.9) Proteins were identified by peptide mass fingerprint and confirmed by MS/MS by MALDI ToF/ToF. * Mascot Score, se- Int. J.quence Mol. Sci.and intensity 2021, 22, 5122 coverage are expressed as representative values. ** Only identified by MS/MS. 7 of 17 2.4. C3 Alternative Pathway Components Expression in Vascular Wall Resident Cells Complement factor C3 was consistently transcribed by human VSMCs (hVSMCs), 2.4. C3 Alternative Pathway Components Expression in Vascular Wall Resident Cells and C3mRNA levels were up-regulated (1.7-fold, p < 0.05; Figure 4A) in lipid-loaded hVSMCs Complement factor C3 (24 h exposure wasµg/mL to 100 consistently transcribed aggregated LDL by humanThe agLDL). VSMCs (hVSMCs), significant and protein C3mRNA levels were up-regulated (1.7-fold, p < 0.05; Figure 4A) expression of C3, receptor C3aR (anaphylatoxin receptor) and integrin αMβ2 in lipid-loaded hVSMCs (24 h exposure receptor (CD11b/CD18) to 100 µg/mL aggregated for C3b/iC3b wasLDL also agLDL). observedThe significant in hVSMCs protein4B). (Figure expression In con- of C3, receptor C3aR (anaphylatoxin receptor) and integrin trast, agLDL did not significantly affect the protein expressionMlevelsα β 2 (CD11b/CD18) receptor of the cell membrane for C3b/iC3b receptors C3aR was also observed and integrin in hVSMCs αMβ2 (Figure (Figure 4B). the 4C). Interestingly, In contrast, agLDL protein levels of did not C3 (C3 significantly affect the protein expression levels of the cell membrane α-chain) were significantly increased in cell culture supernatants when hVSMCs were in- receptors C3aR and integrin cubated in the α M β2 (Figure presence 4C). Interestingly, of agLDL (Figure 4B). Allthe protein these together, levelsresults of C3 indicate (C3 α-chain) were that there significantly increased in cell culture supernatants when hVSMCs were is a local synthesis of C3 components that were released to the ECM of atherosclerotic incubated in the presence of agLDL (Figure 4B). All together, these results indicate that there is a local plaques. synthesis of C3 components that were released to the ECM of atherosclerotic plaques. Figure 4. C3 alternative pathway components expression in human VSMCs. (A) mRNA quantifi- Figure 4. C3 alternative pathway components expression in human VSMCs. (A) mRNA quantifica- cation tion by real-time by real-time PCRPCRusingusing specific specific primers primers for human for human C3 in human C3 in human VSMCs VSMCs treated treated with with or or without without agLDL agLDL (100 (100(B) µg/mL). Protein(B) µg/mL). Protein levels of C3levels of C3 and C3-derived and C3-derived products in products in the supernatant the supernatant of hVSMCs of hVSMCs treated withtreated with oragLDL or without without agLDL (100 (100Human µg/mL). µg/mL).serumHuman serumwas (Serum) (Serum) usedwas as aused as a control positive positive for C3 (nfor control = 3 C3 independent experiments). (n = 3 independent (C) Western experiments). (C)blot analysis Western blotfor C3a receptor analysis for C3a(C3aR) receptor αMβ2 and(C3aR) (C11b/CD18) integrin (receptor and αM β2 (C11b/CD18) for(receptor integrin C3b/iC3b) for in lysates ofinhVSMCs C3b/iC3b) lysates ofincubated with or without hVSMCs incubated with or agLDL without(100 µg/mL). agLDL (100 Band intensity µg/mL). is given in Band intensity is arbitrary units as mean given in arbitrary units as± SEM meanand statistical ± SEM signifi- and statistical cance (p < 0.05, Mann–Whitney test) is indicated (n = 4 independent experiments). significance (p < 0.05, Mann–Whitney test) is indicated (n = 4 independent experiments). 2.5. Exogenous C3 Proteolytic Products, AgLDL and VSMC Function Lipid-loaded VSMCs have an impaired migration rate and cell attachment dynam- ics [29–31]. As shown in Figure 5, exogenously added C3 proteolytic products (10 nM C3a or 100 nM iC3b) partially reversed the impairment of the human VSMC (hVSMC) repair function induced by aggregated LDL (agLDL) to levels that did not differ significantly from the wound repairing capacity of hVSMC control cells. C3a induced a significant increase in the migrating capacity of lipid-loaded hVSMCs into the wound area. A similar trend, although non-significant, was observed with iC3b. C3 proteolytic products did not affect the wound-repairing capacity of control VSMCs (not exposed to agLDL). Moreover, the addition of iC3b (100nM) to hVSMCs in adhesion experiments induced a significant increase in the attachment capacity of the cells, both in the absence and presence of agLDL. The effect was more evident (higher percentage of increase) and more prolonged in time (up to 2 h after seeding) in the lipid-loaded hVSMCs compared to cells not exposed to agLDL (Table 3). In addition, exogenously added iC3b enhanced the organization of the F-actin cytoskeleton during cell adhesion. This was especially evident in hVSMCs exposed 2.5. Exogenous C3 Proteolytic Products, AgLDL and VSMC Function Lipid-loaded VSMCs have an impaired migration rate and cell attachment dynamics [29–31]. As shown in Figure 5, exogenously added C3 proteolytic products (10 nM C3a or Int. J. Mol. Sci. 2021, 22, 5122 100 nM iC3b) partially reversed the impairment of the human VSMC (hVSMC) repair 8 of 17 function induced by aggregated LDL (agLDL) to levels that did not differ significantly from the wound repairing capacity of hVSMC control cells. C3a induced a significant in- crease in the migrating capacity of lipid-loaded hVSMCs into the wound area. A similar to agLDL trend, that otherwise although did not non-significant, show was any organized observed netC3 with iC3b. ofproteolytic actin fibersproducts (F-actin positive), did not 60 min affect theafter seeding (Figure wound-repairing 6). capacity of control VSMCs (not exposed to agLDL). Figure Effects of Figure5.5. Effects of exogenous exogenousC3C3 proteolytic products proteolytic productson migration of human on migration lipid-loaded of human hVSMCs. lipid-loaded hVSMCs. Results for wound coverage (%) in an in vitro model of wound repairing Results for wound coverage (%) in an in vitro model of wound repairing of FCS-stimulated hVSMCs of FCS-stimu- lated hVSMCs treated treated with/without with/without agLDL (100 µg/mL)agLDLin (100 theµg/mL) presenceinorthe presence absence or absence of C3a (10 nM) orof iC3b C3a (10 (100nM) nM), or(niC3b (100 nM), (n = 6 independent experiments in duplicates). (A) Time-course = 6 independent experiments in duplicates). (A) Time-course for wound coverage by hVSMCs. for wound cover- age by hVSMCs. Ctrol and LDL: cells incubated in the absence of C3 products, without or with Ctrol and LDL: cells incubated in the absence of C3 products, without or with agLDL, respectively. agLDL, respectively. iC3b and L+iC3b: cells incubated with iC3b without or with agLDL. C3a and iC3b and L+iC3b: cells incubated with iC3b without or with agLDL. C3a and L+C3a: cells incubated L+C3a: cells incubated with C3a without or with agLDL. (B) Representative microphotographs of with C3a without wound-repairing or with model by agLDL. hVSMCs (B)taken Representative at 0 and 8 hmicrophotographs of wound-repairing after inducing double-side injury. Barmodel dia- by hVSMCs taken at 0 and 8 h after inducing double-side injury. Bar diagrams grams refer to quantitative values for the % of wound covered area after 8 h injury. Band refer to quantitative intensity isvalues given for the % of wound in arbitrary units ascovered mean ±areaSEMafter (n =86hindependent injury. Band experiments intensity is given in arbitraryp units in duplicates). < 0.05as mean was ± SEM (n considered = 6 independent statistically experiments significant in duplicates). (Mann–Whitney p < 0.05 wastests). and Kruskal–Wallis considered statistically significant (Mann–Whitney and Kruskal–Wallis tests). Moreover, the addition of iC3b (100nM) to hVSMCs in adhesion experiments in- Effect of iC3b Table a3.significant duced on cell in increase adhesion capacity incapacity the attachment the absence andcells, of the presence bothofinagLDL. the absence and presence of agLDL. The effect Controlwas more evident (higher percentage +iC3b of increase) and more prolonged in time (up to 2 h after seeding) in the lipid-loaded hVSMCs compared to cells 30 min 60 min 120 min not exposed to agLDL (Table 3). In addition, exogenously added iC3b enhanced the or- −agLDL 100.0 ± 0.0 93.9 ± 4.4 115.9 ± 4.8 * 93.7 ± 14.0 ganization +agLDLof the F-actin100.0 cytoskeleton ± 0.0 during65.4 cell ± 1.8 adhesion.122.0 This ±was2.6especially 177.1evident ± 42.3 *in hVSMCs exposed to agLDL that otherwise did not show any organized net of actin fibersof Results refer to the number of attached cells from a total of 1 × 105 seeded cells, expressed as percentage (F-actin attachedpositive), 60 minnon-receiving cells in the groups after seeding iC3b(Figure (controls).6). Cell viability was in all cases >95% as determined by trypan blue staining. Results are given as mean ± SD of three independent experiments in duplicates. * p-values for comparison (Mann–Whitney test) between cells with/without agLDL −/+ addition of exogenous iC3b at 3 different time points. * p < 0.05. 30 min 60 min 120 min −agLDL 100.0 ± 0.0 93.9 ± 4.4 115.9 ± 4.8 * 93.7 ± 14.0 +agLDL 100.0 ± 0.0 65.4 ± 1.8 122.0 ± 2.6 177.1 ± 42.3 * Results refer to the number of attached cells from a total of 1 × 105 seeded cells, expressed as percentage of attached cells in the Int. J. Mol. Sci.groups 2021, 22,non-receiving 5122 iC3b (controls). Cell viability was in all cases >95% as determined by trypan blue staining. 9 of 17 Results are given as mean ± SD of three independent experiments in duplicates. * P-values for comparison (Mann–Whitney test) between cells with/without agLDL −/+ addition of exogenous iC3b at 3 different time points. * P < 0.05. Figure 6. iC3b induces actin fiber polymerization and cytoskeleton rearrangement in hVSMCs Figure 6.toiC3b exposed induces agLDL. actin fiber Confocal polymerization microscopy images ofand cytoskeleton attached rearrangement hVSMCs, 60 min afterinseeding. hVSMCsRep- exposed to agLDL. Confocal microscopy images of attached hVSMCs, 60 min after seeding. Repre- resentative photomicrographs of control cells and agLDL treated cells, in the presence/absence of sentative photomicrographs of control cells and agLDL treated cells, in the presence/absence of exogenous iC3b (100 nM). Cells were labelled for F-actin with Alexa Cy3 488-phalloidin. exogenous iC3b (100 nM). Cells were labelled for F-actin with Alexa Cy3 488-phalloidin. 3. Discussion 3. Discussion The C3 complement system is an important mediator of innate immunity responses and aThe keyC3component complement of system is an important the complement system. mediator of innate immunity Accumulating evidence responses indicates and high that a keylevels componentof C3 of the complement account system. risk for an increased Accumulating evidencedisease of cardiovascular indicatesin that hu- high [19,22,32] mans levels of C3 accountinfor especially an increased patients risk of pathologies with metabolic cardiovascular [33].disease To date,inhowever, humans [19,22,32] most of theespecially in patients studies addressed towith metabolic investigate the pathologies involvement[33]. To complement of the date, however, most system ofthe in the atherosclerotic studies addressed to investigate process have focused the involvement on the C5b-derived of the complement terminal pathwaysystemof inthe the complement system (reviewed in [14,15]). atherosclerotic process have focused on the C5b-derived terminal pathway of the comple- mentPrevious studies primarily system (reviewed in [14,15]). employed immunohistochemistry to localize C3 compo- nentsPrevious in the human atherosclerotic studies primarily employedvessel wallimmunohistochemistry [18,19]. Here, using mass tospectrometry-based localize C3 compo- proteomics, nents in thewe identified human a differentialvessel atherosclerotic protein signature wall [18,19].ofHere, the alternative using mass C3spectrometry- complement system in the intimawe based proteomics, layer of atherosclerotic identified a differential lesions proteincompared signature to macroscopically of the alternativeapparent C3 com- normal segments of human aortas. It is important to note that the plement system in the intima layer of atherosclerotic lesions compared to macroscopically sequential protein extrac- tion method used in this study made it possible to separately apparent normal segments of human aortas. It is important to note that the sequential investigate proteins located in the extracellular protein matrix (ECM) extraction method used inand thisthose studyin made the cellular fraction it possible of the intima. to separately Using investigate this approach, proteins locatedweinrevealed an enrichment the extracellular matrixof(ECM) the C3and complement those in the proteolytic productsofand cellular fraction the regulators intima. Using of the thisC3approach, cascade, we such as the factor revealed H/CFHRof an enrichment family the C3incomplement the matrisome (subset proteolytic of non-structural products regulatory and regulators ofproteins) of the human the C3 cascade, such atherosclerotic as the factor H/CFHR ECM. family in the In addition to being a structural support to matrisome (subset of non-structural regulatory proteins) of the humanprovide cell anchorage, the components atheroscleroticof the ECM. ECM interact with vascular resident and infiltrated cells, regulating their phenotype and function In addition[34].toThus, beingour results strongly a structural supportsuggest that the to provide cellC3 complement anchorage, thecomponents components present of the ECM interact with vascular resident and infiltrated cells, regulating theirthat in the atherosclerotic ECM are key players in the outside-in signaling occurs phenotype in andkey vascular function cells [34]. involved Thus, our resultsin atherosclerosis strongly suggest progression. that the C3 Incomplement agreement, we and oth- components ers [35,36] have consistently identified anaphylatoxin receptors present in the atherosclerotic ECM are key players in the outside-in signaling that occurs in human atherosclerotic arteries. In addition, in key vascular this study cells involved evidenced increased in atherosclerosis levels of progression. In α M β2 integrin agreement, we inandthe cell others fraction of atherosclerotic lesions, supporting the relevance of the iC3b/C3b-mediated [35,36] have consistently identified anaphylatoxin receptors in human atherosclerotic ar- signaling in the atherosclerotic process. teries. In addition, this study evidenced increased levels of αMβ2 integrin in the cell frac- An unresolved question, with apparently controversial findings to date, refers to tion of atherosclerotic lesions, supporting the relevance of the iC3b/C3b-mediated signal- the origin of the vascular components of the alternative complement system since the ing in the atherosclerotic process. C3 complement is mainly synthesized in the liver [37,38]. In the present study, we evi- An unresolved question, with apparently controversial findings to date, refers to the denced elevated levels of circulating C3 in clinically asymptomatic patients with genetic origin of the vascular components of the alternative complement system since the C3 diagnosis of FH and subclinical atherosclerosis (assessed by CTA) when compared with plasma levels in young healthy subjects, suggesting a maintained activation of the innate immune response in FH, although all patients were long-term treated with LLT as per the guidelines [27]. Interestingly, these results extend our own previous findings report- ing on higher levels of cMVs derived from inflammatory cells, specifically monocyte- and lymphocyte-derived cMVs, in FH patients under long-term LLT [6]. In this respect, monocyte/macrophage has been described to express complement proteins in response to several pro-atherogenic conditions, including high levels of cholesterol [39]. It is worth noting, however, that all FH patients in our study had circulating levels of CRP below 1mg/L, showing very low systemic inflammation which might be related to the fact that all patients were treated according to the guidelines with the highest LLT for more than one Int. J. Mol. Sci. 2021, 22, 5122 10 of 17 year, and they had a well-compensated lipid profile. Further studies might help to gain a better understanding of the association between immune cell activation and inflammatory markers in heterozygous FH patients and their relevance for disease progression. Plasmatic C3 was positively correlated with levels of LDL-C and Apo-B in the whole population under study (healthy subjects and FH patients), whereas no correlation was found with other lipid components, such as triglycerides and HDL-C. In agreement, a previous study based on two-dimensional electrophoresis analysis of serum samples from four FH patients and four healthy subjects identified a protein spot as C3 in the serum of FH patients and reported a positive correlation between levels of the C3a product and total plasma cholesterol [23]. From our results in FH patients, we could not exclude that the higher content of C3 in the atherosclerotic ECM results from the LDL flux into the intimal arterial wall and the retention of ApoB-rich lipoprotein particles in the intimal ECM space by binding to proteoglycans, which in turn favors their modification to form aggregates (agLDL) [40,41]. However, in the present study, although we found a local C3 accumulation in the atherosclerotic ECM of human aortas, the levels of C3 in the systemic circulation do not seem to be a sensitive measure of the plaque burden severity measured by CTA imaging in FH patients with subclinical coronary atherosclerosis. This finding prompts us to hypothesize that vascular resident cells may represent a main source of C3 in human atherosclerotic plaques. Supporting our view, experimental studies on hyperlipidemic Apolipoprotein-E knockout (ApoE-KO) mice fed a high-fat diet evidenced C3mRNA up-regulation in aortic tissue, even before atherosclerotic plaque formation [23]. Interestingly, clonally expand- ing SMC, recently linked to neointimal formation and atherosclerotic plaque pathogene- sis [42,43], overexpressed C3 complement factor in ApoE-/- mice fed a high-fat Western diet [22]. Due to the post-mortem condition of the aortic samples, we could not ana- lyze mRNA expression in the human atherosclerotic lesions in the present study, but we evidenced that cultured human VSMCs express C3mRNA and that C3 complement is consistently over-expressed when cells are exposed to atherogenic agLDL for 24 h periods. In agreement with this, supernatants of lipid-loaded cells were enriched in the C3α chain, a C3 form that was not present in the agLDL, but significantly increased in the atherosclerotic ECM of human aortas, supporting the relevance of VSMCs as a source of C3 complement active forms in the lesions. In contrast, the C3c fragments detected in the atherosclerotic intimal ECM seem mainly to have a systemic origin, entering the arterial intima through the LDL. Indeed, the serum showed a strong signal for C3c fragments, the final proteolysis product derived from C3 complement activation, when analyzed by Western blot, and a similar fragment was also found in purified agLDL (Figure S2). C3c has been described as a biomarker of heart failure [44], periodontitis [45] or amyotrophic lateral sclerosis [46], but its role and function needs to be further investigated. As shown by Wang et al., C3 expressed by the clonal SMC induced proatherogenic effects, including the paracrine regulation of macrophage inflammation and autocrine- induced SMC proliferation [22]. Among the C3 proteolytic fragments resulting from the C3 cascade activation, C3a and iC3b/C3b are widely recognized as highly bioactive molecules directly involved in the regulation of cell phenotype and function [47]. In particular, C3a has been described to act as a chemoattractant for neural crest cells [48] and, through its receptor C3aR, to control leukocyte recruitment and endothelial activation in cerebral microvessel inflammation [49]. We previously described that agLDL, resembling the LDL retained and aggregated in the ECM of the intimal layer in areas with atherosclerosis [40,50,51], is internalized by human VSMCs, inducing changes in their phenotype and impairing cell functions, such as adhesion and migration, mainly mediated by effects on actin-cytoskeleton dynamics and organization [31,52,53]. In the present study, we demonstrate that the inhibitory effect of agLDL on VSMC migration is ameliorated by the presence of exogenous C3a to a level that did not significantly differ from the migration capacity of the control group (non-exposed to agLDL) in an in vitro wound healing assay. Interestingly, C3a did not Int. J. Mol. Sci. 2021, 22, 5122 11 of 17 show a similar enhancing effect of cell migration in the control hVSMCs. Similarly, we found that exogenously added- iC3b promoted attachment during the cell adhesion and reorganization of the F-actin cytoskeleton network. This effect was more evident and maintained in lipid-loaded cells that otherwise would not present any organized actin cytoskeleton shortly after seeding. In summary, our results demonstrated for the first time the presence and differential abundance of active products of the C3 system in the ECM of human atherosclerotic lesions. In addition, we provided evidence of the capacity of C3-derived products, beyond their well-known role in inflammation and immunity, to modulate the migratory and repair function of VSMCs that is impaired by LDL. These results suggest the C3 complement pathway is a novel player in vascular remodeling and in the progression of advanced human atherosclerotic lesions. 4. Materials and Methods 4.1. Human Samples 4.1.1. Subjects with Familial Hypercholesterolemia and Healthy Volunteers The present study included 49 subjects with a genetic diagnosis of heterozygous familial hypercholesterolemia (FH) and thus lifelong exposure to high LDL plasma levels and high risk of premature atherosclerosis from the SAFEHEART cohort. A group of young healthy volunteers (non-FH subjects, N = 28) from the same cohort was used as reference group to establish the normal plasma range of C3 levels in a healthy population, at very low risk of presenting subclinical atherosclerosis. Demographic and clinical data of the FH patients and the healthy volunteers are provided in Table 1 and Table S1. Neither the FH nor the healthy group included pregnant subjects. Cases of sepsis or infections and with history of cancer or suspected clinical cardiovascular events were excluded. This part of the study was approved by the Local Ethics Committee for Clinical Investigation in the Fundación Jimenez Diaz (CEIC-FJD; Madrid, Spain) (protocol number: 01/09) and was conducted according to the Declaration of Helsinki (2013), and written informed consent was obtained from all participants [54]. Coronary atherosclerotic plaque characterization was performed by computed tomo- graphic angiography (CTA), as previously described [27]. Coronary atherosclerotic-plaque burden was characterized and quantified using the SAPC (QAngio CT (Research Edition V2.1.16.1; Medis Specials, Leiden, The Netherlands) software. SAPC measurements were performed by a blinded operator, unaware of any clinical or biochemical data. [27]. 4.1.2. Aortas and Coronary Arteries Abdominal aortic tissue was obtained from the Biobank of the Eulalia Study on out-of-hospital sudden death [28]. Autopsy was performed within 18 h after death (age 34–79 years old) following the established forensic protocol [55,56], and the study was approved by the Institutional Ethical Committee for Clinical Investigation (Hospital Santa Creu i Sant Pau; Barcelona, Spain). The samples were processed immediately. After the removal of connective tissue and adherent blood, the specimens were divided into grossly homogeneous parts. Aortic wall segments were classified by their macroscopic appearance according to the presence and severity of atherosclerotic lesions. In this study, we compared macroscopically normal-appearing areas with atherosclerotic plaques (raised white or yellow-white plaques) obtained from the same artery (N = 4 individual aortas). From all segments, the intima layer was dissected from the media, snap-frozen in liquid N2 and stored at −80 ◦ C. To confirm the validity of the macroscopic classification, representative samples of each type of segment were examined histologically. To this aim, segments from each aortic tissue were embedded with paraffin, and 5µM sections were stained with Massons’s trichromic to identify cellular areas (Figure S3). The images were captured with an Olympus microscope Vanox AHBT3 (Hamburg, Germany) coupled with a Sony 3CCD color video camera and processed using Visilog (Sony ESPAC, San Jose, CA, USA) software (version 4.1.5). Int. J. Mol. Sci. 2021, 22, 5122 12 of 17 4.1.3. VSMC Culture and LDL Preparation Primary human VSMCs (Cell Application, Inc., San Diego, CA, USA) were cultured in M199 medium containing 20% FBS and used between passage four and seven, as previously described [30]. Unless otherwise indicated, experiments were performed in subconfluent monolayers after incubation without or with aggregated LDL (agLDL; 100 µg/mL) for 16 h. Human LDLs (density 1.019–1.063 g/mL) were purified by ultracentrifugation from pooled sera of normocholesterolemic volunteers, and agLDLs were generated by vortexing LDL (1 mg/mL), according to the initial method described by Guyton et al. [57] and as previously performed in our group [58]. This method has been shown to produce similar LDL aggregation as LDL versican incubation [59]. LDL protein concentration was determined using the bicinchoninic acid (BCA)- method (ThermoFisher, Rockford, IL, USA) and LDL purity assessed by agarose gel electrophoresis (SAS-MX Lipo-kit, Helena Biosciences, Gateheads, UK). LDL prepara- tions were tested to exclude the presence of endotoxin (Limulus amebocyte lysate test, BioWhittaker, Walkersville, MD, USA) and potential bacterial contamination that could derive in confounding results, and this proved to be negative in all cases. LDLs used in the experiments were less than 48 h old. LDL oxidation in all LDL preparations was excluded by assessing thiobarbituric-acid-reactive substance (TBARS) formation, according to Ohkawa et al. [60] with slight modifications [61]. 4.2. Tissue Processing and Extraction of ECM Proteins Aortic ECM proteins were extracted according to Didangelos et al. [62]. Briefly, 300 mg segments of aortic tissue (intima layer) were diced in 8–10 pieces (approximately 2 × 2 mm2 size) and washed 5 times with PBS containing 25 mM EDTA. ECM-soluble proteins were obtained by incubating the aortic samples for 4 h at room temperature (RT) under mild agitation at 800 rpm, with 0.5 M NaCl, 10 mM Tris (pH 7.5), supplemented with 25 mM EDTA (10:1 buffer volume to tissue weight). Tissue pieces were left to drop. Then, the supernatant was collected, cleaned up with desalting Zeba-Spin columns (ThermoFisher, Rockford, IL, USA) and precipitated overnight with 5 volumes of chilled 100% acetone at −20 ◦ C. Proteins were re-dissolved in deglycosilation buffer (NaCl 150 mM; sodium acetate 50 mM; EDTA 10 mM, supplemented with 0.05 units of HeparinaseII; Chondroitinase ABC and Endo-β-Galactosidase). Then, tissue samples were incubated with 0.08% SDS (10:1 buffer volume to tissue weight) and 25 mM EDTA for a further 4 h (RT, mild agitation at 800 rpm). SDS supernatant was collected. Thereafter, tissue pieces were incubated for 48 h in guanidine-HCl buffer (4 M guanidine-HCl, 50 mM sodium acetate, pH 5.8 supplemented with 25 mM EDTA). After removing the guanidine with 100% ethanol, samples were centrifuged at 16,000× g (10 min) and stored at −80 ◦ C. All buffers contained protease inhibitors (1 tablet/50 mL, Complete-EDTA free Roche) and phosphatase inhibitors (1%). Reagents were obtained from Sigma-Aldrich ((Merck KGaA, Darmstadt, Germany). The purity of each fraction was confirmed by Western blot with specific antibodies against β-actin (ab8228, Abcam, 1/1000); ColI (ab6308, Abcam, 1/1000) and AEBP1 (#250461, Antibodies Online, 1/500; Aachen, Germany), that specifically partitioned in the NaCl-, SDS- and guanidin-HCl-fractions (Figure S4). 4.3. 2D Electrophoresis/Mass Spectrometry Analysis Proteins (150 µg) were identified by matrix-assisted laser desorption/ionization time of flight (MALDI-ToF/ToF) mass spectrometry (Bruker Daltonics Autoflex III Smartbeam; Bruker Daltonik GmbH, Leipzig, Germany) after separation by two-dimensional elec- trophoresis (2-DE) [63]. Differential protein pattern analysis by 2DE was performed with arterial segments (apparently normal and atherosclerotic) obtained from 3 independent aortas. To guarantee the highest homogeneity and ensure better comparability, protein ex- tracts from all arterial segments (N = 6) were run simultaneously in an Ettan-Dalt-6 Device (GE-Healthcare, Uppsala, Sweden), and analysis was performed in duplicate. Protein spots Int. J. Mol. Sci. 2021, 22, 5122 13 of 17 in the gels were labeled by fluorescence (Flamingo labeling, Bio-Rad), scanned (Typhoon, GE-Healthcare, Uppsala, Sweden) and analyzed for differences in the protein pattern be- tween groups with PD-Quest 8.0.1 software (Bio-Rad, Hercules, CA, USA). Quantification of the protein spot volume was performed with the PD-Quest 8.0.1 software (Bio-Rad, Hercules, CA, USA) as previously described [29,63]. Briefly, the software created a single master that included all gels included in the analysis. A relative value that corresponds to the single spot volume compared to the total volume of the spots in each gel was assigned to each spot in the gels. Afterwards, this value was subjected to background extraction, and the final intensity value was then normalized by the local regression model (LOESS) method included in the software [29,63]. For protein identification, mass spectrometry (MS) spectra were submitted to a MASCOT (Matrix Science Ltd, London, UK) search on Swiss-Prot 57.15 database using the following parameters: taxonomy Homo sapiens, mass tolerance 50–100, up to 2 missed cleavages; carbamidomethyl (C) as global modification and oxidation (M) as variable modification. Identification was accepted with a score higher than 56 for peptide mass fingerprint and 20 for MS/MS. 4.4. Western Blot and ELISA Assays Protein antigen levels in total lysates, obtained with RIPA buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 0.5% triton X-100, 0.5% Sodium deoxyclodate, 0.1% SDS) as we previously described [30], and from ECM extracts (see above), were analyzed by Western blot, as described previously [52] using the following primary antibodies: C3 (ab200199, dilution 1/2000, Abcam, Cambridge, UK ); C5 (Abcam ab11876, dilution 1/500); C3aR (Abcam ab126250, dilution 1/1000); CD11b (Abcam ab133357, dilution 1/1000); CD18 (Abcam ab119830, dilution 1/500); Human β-actin (Abcam ab8226, dilution 1/5000) and total protein (Ponceau staining) were used as loading controls. Western blot bands were visualized by chemiluminescence using a peroxidase enzymatic reaction (Supersignal, ThermoFisher, Rockford, IL, USA)) and quantified with a ChemiDoc™ XRS system using Image Lab software (Bio-Rad, Hercules, CA, USA). Quantitative plasma analysis of C3 was performed by a commercial double antibody sandwich enzyme-linked immunosorbent assay (AssayPro EC2101-1, St Charles MO, USA) with a lower limit of detection of 83 ng/mL calculated by 2SD from the mean of a zero standard. The intra-assay and inter-assay precision were CV < 5.2 and <8.9%, respectively. 4.5. RNA Extraction and Real-Time PCR Analysis Total RNA was extracted from areas with migrating VSMCs (wound border) or non- migrating cells after 6 h of wounding (Figure S3) or from growth-arrested cells (maintained 18 h in M199 without FBS supplementation) using an RNeasy Mini Kit (Qiagen, ref. 74104), according to the manufacturer’s instructions. RNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and purity was checked with the A260/A280 ratio. mRNA levels were analyzed by real-time PCR [59] using an RT2 Profiler PCR targeted array for human cell motility (Qiagen; Cat. no. 330231 PAHS-128ZA) to compare gene expression profiles between migrating and non-migrating hVSMCs and with TaqMan fluorescent real-time PCR probes (ThermoFischer, Rockford, IL, USA)) to quantify C3 (Hs00163811-m1). Human GAPDH (4326317E) was used as an endogenous control. Sam- ples were analyzed in duplicate, and only mRNAs with expression levels below 32 cycles were accepted. 4.6. Cell Adhesion and Wound-Healing Assays Migration studies were performed with human VSMCs seeded in a culture insert (ibidi 2-well culture insert, ibidi GmbH, Martinsried, Germany) and left in M-199 with 10% FCS with or without 100 µg/mL agLDL until cell confluence was achieved. When indicated, C3a (10 nM) or iC3b (100 nM) were added to the culture medium 1 h before stimulation with agLDL and maintained during the assay. After removing the culture Int. J. Mol. Sci. 2021, 22, 5122 14 of 17 inserts, cells were washed with PBS and maintained in M199 migration medium (10% FCS) with its corresponding treatment for a total of 8 h. Migration on the cell-depleted area was controlled using an inverted microscope (Leica DMIRE2, Wetzlar, Germay) with a 10× lens magnification. Images were taken at 2 h intervals. During migration, cells were maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . The cell-free area of each field was quantitatively determined using ImageJ software. Changes in the viability of the cells due to agLDL had been previously excluded [64]. Cell attachment studies were performed as previously described [53]. Briefly, sub- confluent cultures of VSMCs were incubated with or without iC3b (100 nM), in the pres- ence/absence of agLDL (100 µg/mL) for 16 h. Cells were then harvested with trypsin, suspended in 5% FBS-containing medium and seeded (1 × 105 cells) on FBS-coated glass bottom dishes in the presence or absence of iC3b (100 nM) and/or agLDL (100 µg/mL). At different time periods (30 min, 1 h and 3 h), attached cells were released by trypsination, stained with trypan blue for determination of cell viability and counted in a Neubauer chamber. Alternatively, at these time periods, cells were fixed with 4% paraformaldehyde for immunolabeling and confocal microscopy. 4.7. Confocal Focal Microscopy Cells fixed with 4% paraformaldehyde were permeabilized (0.5% Tween-PBS), blocked with 1% bovine serum albumin (BSA), immunolabeled for F-actin and analyzed by confocal microscopy as previously described [30,31,52] using Alexa Fluor 633 or 488 phalloidin (Molecular Probes) on a Leica TCS SP2-AOBS inverted fluorescence microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany). Fluorescent images were acquired in a scan format of 1024 × 1024 pixels at intervals of 0.1 mm (20 slides) and processed with the TCS-AOBS software (Leica). Maximal intensity projection values were calculated using the LASAF Leica Software and given as AU/mm2 . 4.8. Statistical Analysis Results are presented as the mean ± SEM (standard error of the mean), except when indicated. Outlier expressions were excluded by Chauvenet’s criterion. Sample distribution was verified by the Shapiro–Wilk test. Statistical differences between groups were analyzed by non-parametric tests (Kruskal–Wallis or Mann–Whitney), as indicated. StatView soft- ware (Abacus Concepts) and SPSS Statistics Version 21.0.0 (SPSS, Chicago, IL, USA) were used for statistical analysis, and a p-value < 0.05 was considered statistically significant. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22105122/s1. Author Contributions: Conceptualization, T.P. and L.B.; methodology, M.G.-A. and E.P.; validation, M.G.-A., E.D.-R. and T.P.; formal analysis, T.P., L.B., M.G.-A., E.D.-R. and R.E.; investigation, M.G.-A., E.D.-R., E.P., R.E., P.M., O.J.-B. and T.P.; resources, L.B. and T.P.; writing—original draft preparation, M.G.-A., T.P. and L.B.; writing—review and editing, M.G.-A., E.D.-R., E.P., R.E., O.J.-B., P.M., L.B., T.P.; visualization, M.G.-A., L.B., T.P.; supervision, T.P. and L.B.; project administration, T.P. and L.B.; funding acquisition, T.P. and L.B. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Institute of Health Carlos III, ISCIII [FIS PI19/01687, to T.P.; Red Terapia Celular TerCel-RD16/0011/0018 to L.B.; CIBERCV to L.B.; Spanish Ministry of Economy and Competitiveness of Science-PID2019-107160RB-I00 to L.B.; and cofounded by FEDER “Una Manera de Hacer Europa”. Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya [2017 SGR 1480]. We thank Fundación Jesus Serra and Fundación de Investigación Cardiovascular, Barcelona, for their continuous support. Institutional Review Board Statement: The study in humans (SAFEHEART Cohort) was approved by the Local Ethics Committee of the Investigación Clínica Fundación Jimenez Diaz (CEIC-FJD; Madrid, Spain) (protocol number: 01/09) and was conducted according to the Declaration of Helsinki (2013). Post-mortem studies in autopsy material were performed following an established forensic Int. J. Mol. Sci. 2021, 22, 5122 15 of 17 protocol [56,57], and the study was approved by the Institutional Ethical Committee for Clinical Investigation of the Hospital Santa Creu i Sant Pau (Barcelona, Spain). Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not Applicable. Acknowledgments: Authors are indebted to Roberta Lugano, Montse Gomez-Pardo and Esther Gerbolès for their technical support. R.E. is a CIBERCV investigator. E.DR. is a recipient of a predoctoral research fellowship from the Cardiovascular Program-ICCC (IR-HSCSP). Conflicts of Interest: The authors declare no conflict of interest. References 1. Ridker, P.M. LDL cholesterol: Controversies and future therapeutic directions. Lancet 2014, 384, 607–617. [CrossRef] 2. Vogt, A. The genetics of familial hypercholesterolemia and emerging therapies. 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