Deletion or Inhibition of NOD1 Favors Plaque Stability and Attenuates Atherothrombosis in Advanced Atherogenesis

cells Article Deletion or Inhibition of NOD1 Favors Plaque Stability and Attenuates Atherothrombosis in Advanced Atherogenesis Silvia González-Ramos 1,2,*,†, Victoria Fernández-García1,2,†, Miriam Recalde 1, Cristina Rodríguez 2,3 , JoséMartínez-González 2,4 , Vicente Andrés2,5 , Paloma Martín-Sanz 1,6 and Lisardo Boscá1,2,* 1Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), 28029 Madrid, Spain; [email protected] (V.F.-G.); [email protected] (M.R.); [email protected] (P.M.-S.) 2Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain; [email protected] (C.R.); [email protected] (J.M.-G.); [email protected] (V.A.) 3Institut de Recerca del Hospital de la Santa Creu i Sant Pau-Programa ICCC, IIB Sant Pau, 08041 Barcelona, Spain 4Instituto de Investigaciones Biomédicas de Barcelona (IIBB-CSIC), IIB Sant Pau, 08041 Barcelona, Spain 5Laboratory of Molecular and Genetic Cardiovascular Pathophysiology, Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), 28029 Madrid, Spain 6Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain *Correspondence: [email protected] (S.G.-R.); [email protected] (L.B.); Tel.: +34-(0)91-497-2747 (ext. 5345) (L.B.) †These authors contributed equally to this work. Received: 4 August 2020; Accepted: 9 September 2020; Published: 10 September 2020   Abstract: Atherothrombosis, the main cause of acute coronary syndromes (ACS), is characterized by the rupture of the atherosclerotic plaque followed by the formation of thrombi. Fatal plaque rupture sites show large necrotic cores combined with high levels of inflammation and thin layers of collagen. Plaque necrosis due to the death of macrophages and smooth muscle cells (SMCs) remains critical in the process. To determine the contribution of the innate immunity receptor NOD1 to the stability of atherosclerotic plaque, Apoe −/− and Apoe −/− Nod1 −/− atherosclerosis prone mice were placed on a high-fat diet for 16 weeks to assess post-mortem advanced atherosclerosis in the aortic sinus. The proliferation and apoptosis activity were analyzed, as well as the foam cell formation capacity in these lesions and in primary cultures of macrophages and vascular SMCs obtained from both groups of mice. Our results reinforce the preeminent role for NOD1 in human atherosclerosis. Advanced plaque analysis in the Apoe −/− atherosclerosis model suggests that NOD1 deficiency may decrease the risk of atherothrombosis by decreasing leukocyte infiltration and reducing macrophage apoptosis. Furthermore, Nod1 −/− SMCs exhibit higher proliferation rates and decreased apoptotic activity, contributing to thicker fibrous caps with reduced content of pro-thrombotic collagen. These findings demonstrate a direct link between NOD1 and plaque vulnerability through effects on both macrophages and SMCs, suggesting promising insights for early detection of biomarkers for treating patients before ACS occurs. Keywords: coronary disease; atherothrombosis; vulnerable plaque; innate immunity; pattern recognition receptors Cells 2020,9, 2067; doi:10.3390/cells9092067 www.mdpi.com/journal/cells Cells 2020,9, 2067 2 of 14 1. Introduction According to the World Health Organization, acute coronary syndromes (ACS) are the leading cause of morbidity and mortality in Western society. Rupture of the atheroma underlies most of the thrombotic events that manifest clinically as coronary artery disease, stroke, transient ischemic attack, and peripheral arterial disease [ 1 – 3 ]. Acute coronary events mediated by thrombi depend mainly on the composition and vulnerability of the plaque. The trapping of cholesterol-loaded foam cells [ 4 ] causes thinning of the fibrous cap (FC) and expansion of the necrotic core (NC), as a result of the combination of vascular smooth muscle cells (SMC) and macrophages proliferation and excessive cell death [ 5 , 6 ]. In combination with degradation of the extracellular matrix by proteases released from apoptotic foam cells, the FC is destabilized, leading to its breakdown or erosion, and subsequent exposure to the bloodstream of NC content. As a result, platelets and fibrin aggregate to form a thrombus resulting in partial or total ischemic blockage of the artery [ 7 , 8 ]. Therefore, the clinical atherosclerotic lesion is essentially an unresolved inflammatory condition leading to a vulnerable plaque [ 9 , 10 ]. Notably, atherosclerosis is strongly associated with systemic risk factors (e.g., high LDL, infections, diabetes) where the cellular components of the innate immune system are relevant. In this regard, the nucleotide-binding oligomerization domain (NOD)-1 receptor of the innate immune system appears to play a key role. Our group has recently identified endothelial NOD1 as proatherogenic in response to oxLDL and a conserved region of bacterial peptidoglycan iE-DAP ( γ -D-glutamyl-meso-diaminopimelic acid) [ 9 ]. The downstream interaction of NOD1 with receptor-interacting protein-2 (RIP2 or RICK) activates the canonical nuclear factor kappa B (NFκ B) signaling pathway [ 11 ] that upregulates the expression of adhesion molecules of endothelial cells and the concomitant recruitment of monocytes and neutrophils to the vasculature. Although interest in the study of NOD1 in cardiovascular diseases has increased lately, [ 12 – 15 ] its role in destabilizing atherosclerotic plaque is still lacking. In this study, our aim is to investigate the contribution of NOD1 to key characteristics of plaque vulnerability. We show that NOD1 is induced in SMC and macrophages in human atherosclerotic tissues. Furthermore, the inactivation of Nod1 in the Apoe −/− mouse model of atherosclerosis may contribute to plaque stability by modulating the pathophysiological functions of macrophages and SMCs. 2. Materials and Methods 2.1. Human Samples Human coronary arteries were collected from patients undergoing heart transplant at the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain). Atherosclerotic and non-atherosclerotic coronary arteries were taken from coronary artery disease (CAD) and non-CAD patients, respectively. Written consent was obtained from all participating subjects. The studies were approved by the Ethics Committee of the hospital and were conducted in accordance with the Helsinki Declaration (project RTI2018-094727-B-100; approved October 2018). 2.2. Animal Procedures Animal studies were approved by the local ethics committee, and all animal procedures conformed to EU Directive 2010/63 and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 53/2013. C57BL/6 (Wt) and Apoe −/− mice were obtained from Charles River (JAX mice stock #000664 and #002052, respectively. Barcelona, Spain). Double-knockout Apoe −/− Nod1 −/− mice were generated by crossing Apoe −/− mice with Nod1 −/− mice as previously described [ 9 ]. All experiments compared male Apoe −/− mice vs. male Apoe −/− Nod1 −/− littermates. In order to accelerate the development of atherosclerotic lesions, at 8 weeks of age, males were placed on high-fat diet (HFD, 10.2% hydrogenated coconut oil, 0.75% cholesterol; Ssniff, Soest, Germany) for 16 weeks. Mice were anaesthetized intraperitoneally Cells 2020,9, 2067 3 of 14 under general anesthesia (ketamine/xylazine combination at 80 mg/kg and 10 mg/kg body weight, respectively) before euthanasia by CO2inhalation. Whole blood was extracted post-mortem by cardiac puncture and plasma was obtained by centrifugation at 2000 × gfor 10 min at 4 ◦ C. Plasma concentrations of total cholesterol, free cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were measured enzymatically using kinetic colorimetric kits (Spinreact, St Esteve de Bas, Girona, Spain) according to manufacturer’s instructions. 2.3. Cell Procedures Smooth muscle cells (SMC) were harvested from abdominal and thoracic aortas from 2-month-old animals as previously described [ 16 ] and cultured in Dulbecco’s modified Eagle medium (DMEM, GIBCO, Madrid, Spain) containing 20% fetal bovine serum (FBS, Lonza, Barcelona, Spain), L-glutamine and antibiotics (100 units/mL penicillin and 100 µ g/mL streptomycin). Bone marrow-derived macrophages (BMDM) were obtained from femoral bone marrow suspensions [ 9 ] differentiated for 7 days in the presence of DMEM plus 10% FBS and 20 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech, London, UK). For in vitro apoptosis studies, cells were either irradiated with ultraviolet (UV) light (BMDM: 80 J/m 2 , SMC: 120 J/m 2 ) and cultured an additional 24 h (BMDM) or 48 h (SMC) period, or incubated with the nitric oxide donor S-nitrosoglutathione (GSNO, 1 mM, Sigma, Madrid, Spain); with oxidized LDL (oxLDL, 50 µ g/mL, Biochemistry-Research Unit at Instituto Ram ó n y Cajal de Investigaci ó n Sanitaria, Madrid, Spain); with the NOD1 agonist c12-iE-DAP (1 µ g/mL, Invivogen, San Diego, CA, USA) or with inactive analogue iE-Lys, for 24 h in BMDM cultures or 48 h in SMC cultures. For apoptosis analysis by flow cytometry, control and treated cells were collected and labelled with propidium iodide as per manufacturer’s instructions (ThermoFischer Sci., Madrid, Spain) or processed for cleaved caspase-3 staining as follows: cells were fixed with 1% paraformaldehyde, permeabilized with 0.1% Triton-X100, and stained with a rabbit polyclonal anti-cleaved caspase 3 antibody (1/600, Cell Signaling Technology, Danvers, MA, USA), followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibody (1/500, Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained with DAPI (Life Technologies). Flow cytometry was conducted in a FACSCanto II and DNA histograms were fitted into cell-cycle distributions using the BD FACSDiva software (Beckton Dickinson, Madrid, Spain ). Apoptotic cells were identified as the sub-G0 peak subpopulation after propidium iodide staining or as cleaved caspase-3-positive cells. For SMC, FACS-based cell-cycle analysis, cells were synchronized in G0/G1 by 72 h serum deprivation (DMEM plus 0.1% FBS) and then restimulated with DMEM supplemented with 20% FBS for 12 h and 24 h. Cells were labelled with propidium iodide as described earlier, and processed to fit into same cell-cycle distributions as for FACS-based apoptosis analysis. For NOD1 signaling cascade analysis in SMC or BMDM, cells were pre-incubated when indicated with the NOD1 antagonist Nodinitib-1 (1 µ M, Cayman, Madrid, Spain) for one hour and then treated with LDL (50 or 30 µ g/mL), oxidized LDL (20 or 50 µ g/mL), iE-Lys (a structural analogue of c12-iE-DAP that does not activate NOD1) or the NOD1 activator c12-iE-DAP (1 µg/mL, Invivogen) for 24 h. To detect foam cell formation, BMDM and SMC were incubated with 50 µ g/mL LDL or oxLDL for 24 h and 48 h, respectively, fixed in 1% paraformaldehyde and stained with 0.5% Oil red O (ORO, Sigma) in isopropanol and counterstained with hematoxylin. 2.4. Histological Analysis and Lesion Quantification After mouse cardiac perfusion with PBS supplemented with 5 mM of EDTA, mouse hearts were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h at 4 ◦ C, incubated 24 h in PBS supplemented with 30% sucrose, embedded in OCT and cryopreserved at −70 ◦C. Cryocut cross-sections (5 µ m) of aortic roots were evaluated for conventional hematoxylin-eosin (HE) staining, 0.1% sirius red to detect collagen or 0.5% ORO to detect neutral lipids. Images were captured with a Zeiss Axiophot microscope with a Plan-NEOFLUAR 10x/0.3 objective (Zeiss, Cells 2020,9, 2067 4 of 14 Oberkochen, Germany) and a DP70 camera (Olympus, Madrid, Spain). Polarized images were obtained using a Mirax digital slide scanner (3DHistech, Budapest, Hungary). To avoid specific biases due to potential differences in lesion shape, cross sections of the entire lesion were analyzed and averaged [ 17 ]. To obtain the aortas for the analysis, after fixing in PFA overnight at 4 ◦ C, the aortas were whole-mount stained with 0.2% ORO in methanol, opened longitudinally and pinned to black wax to expose the entire luminal surface. Images were acquired using a Leica MZ6 SZX10 stereomicroscope (Leica Microsystems, Wetzlar, Germany) coupled to a Leica DFC300 digital color camera (Leica Microsystems). The planimetric area of atherosclerotic plaques was measured in pixels using ImageJ. 2.5. Immunostaining Human arteries were fixed overnight in 4% PFA/0.1 M PBS (pH 7.4), embedded in paraffin and sectioned into 5 µ m sections with a microtome (Jung RM2055, Leica). Consecutive deparaffinized sections were rehydrated, subjected to antigen retrieval in 10 mM citrate buffer (pH 6.0), blocked and incubated with a rabbit polyclonal antibody against NOD1 (1:40, Abcam, Cambridge, UK), with a mouse monoclonal anti-smooth muscle α -actin (SMA) alkaline phosphatase-conjugated antibody (1:200, Sigma) or with a rat monoclonal antibody against anti-Mac3 (1:100, Santa Cruz, Santa Cruz, CA, USA). After extensive washes, sections were incubated with correspondingly biotinylated goat anti-rabbit or goat anti-rat secondary antibodies (Vector). Immunocomplexes were detected after incubation with Vectastain Elite ABC reagent (PK6100, Vector, Barcelona, Spain) and DAB substrate (Roche). Images were acquired with an Olympus Vanox AHBT3 microscope and digitalized by a Sony camera (DXC-S500). For immunostaining of cryo-section samples, slides were stained with antibodies specific for mouse Mac3 (1:200, Becton Dickinson), Ly6g (1:100, Becton Dickinson), cleaved caspase 3 (1/600, Cell Signaling Technology) and Ki-67 (1:200, Abcam), followed by secondary staining using standard procedures. Secondary antibodies for immunofluorescence were Alexa Fluor 647-conjugated anti-rabbit (Invitrogen) and Alexa Fluor 594-conjugated anti-rat (Invitrogen). SMC were identified with mouse anti-smooth muscle α -actin FITC-conjugated antibody (1:1000, Sigma). Nuclei were counterstained with DAPI (Life Technologies, Madrid, Spain). Immunofluorescence staining of cryo-sections were mounted in Prolong Gold Antifade mounting medium (Life Technologies). Primary control panel was performed with an appropriate isotype control IgG and secondary controls incubations were performed in the absence of primary antibody. A LSM710 confocal microscope with a Plan-APOCHROMAT 25x/0.8 oil immersion objective (Zeiss) was used to capture images from immunofluorescence staining. Images were analyzed using ImageJ and were processed for presentation with Zen2009 software. 2.6. Western Blot Analysis After stimulation of primary cultures, cells were washed twice with ice-cold PBS. Whole protein extracts were obtained using ice-cold proprietary detergent in 25 mM bicine, 150 mM NaCl; pH 7.6 (T-PER ® Tissue Protein Extraction Reagent, Thermo Fisher Sci.) supplemented with phosphatase cocktail and protease inhibitors (Sigma) [5,18]. Proteins were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes. Proteins were detected using rabbit polyclonal antibody against NOD1 (1:500, Abcam), rabbit polyclonal antibody against phospho-RIP2 (1:1000, Cell Signaling), rabbit polyclonal antibody against RIP2 (1:1000, Cell Signaling), rabbit polyclonal antibody against phospho-NFκ B p65 (1:3000, Santa Cruz), mouse monoclonal antibody against NFκ B p65 (1:1000, Santa Cruz), mouse monoclonal antibody against β-actin (1: 40,000, Sigma), and HRP-conjugated secondary antibodies (BioRad, Hercules, CA, USA). Protein bands were visualized using a Luminata chemiluminescence detection system (Merck Millipore, Madrid, Spain) and a ImageQuant LAS 500 imager (GE Healthcare Life Sci., Madrid, Spain) and were quantified using ImageJ (National Institutes of Health). Protein band intensities of interest were expressed as a percentage of those of the β-actin bands as indicated. Cells 2020,9, 2067 5 of 14 2.7. qRT-PCR Total RNA was isolated by homogenization in QUIAZOL ® by a TissueLyser LT and eluted using MinElute columns (Qiagen; Madrid, Spain). RNA integrity was assessed by RNA Nano Chip (Agilent Technologies; Madrid, Spain). 250 ng of RNA were retro-transcribed by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Madrid, Spain). SYBR Green assay was conducted in 7900HT Fast Real-Time PCR System equipment for qRT-PCR detection of Cd36 (5 0 -AGATGACGTGGCAAAGAACAG-3 0 and 5 0 -CCTTGGCTAGATAACGAACTCTG-3 0 ), Sr-a (5 0 -GAGCCTCGTTCACAGGAGTC-3 0 and 5 0 -CACCAGCTCTAGCATGTCCTC-3 0 ) and Rplp0 (50-ACTGGTCTAGGACCCGAGAAG-30and 50-TCCCACCTTGTCTCCAGTCT-30). Calculations were made from measurement of technical triplicates of each sample. The relative amount of mRNA was calculated with the comparative 2- ∆∆ Ct method using mouse 18S or 36b4 as endogenous control transcripts. 2.8. Quantification and Statistical Analysis All values are expressed as means ± s.e.m. Statistical calculations were performed using GraphPad Prism 6 (GraphPad Software Inc.; San Diego, CA, USA). After calculating for normality by D’Agostino–Pearson omnibus test, either a non-parametric test (Mann–Whitney U-test), or a normality test (unpaired Student’s t test with Welch’s correction) was used as appropriate. Statistical significance was deemed at pvalues <0.05. Removal of outliers was assessed by ROUT method. Statistical tests and pvalues are specified for each panel in the respective figure legends. nindicated in the figure legends refers to the number of individual animals for in vivo and ex vivo assays. 3. Results 3.1. NOD1 in Vascular Smooth Muscle Cells and Macrophages Plays a Key Role in Murine and Human Atherosclerosis Plaque Formation To investigate the role of NOD1 in the advanced stages of atherosclerosis, Apoe −/− Nod1 −/− mice and Apoe −/− controls were placed in a HFD for 16 weeks. Although no significant differences were found between the groups in the lesion area of the aortic valve (Figure 1a), the planimetric analysis of the aorta stained with ORO showed an approximate reduction of 15% in the atheroma of the aortic arch in Apoe −/− Nod1 −/− mice compared to controls (Figure 1b), thus confirming our previous results on the preventive role of Nod1 deletion in early atherosclerosis [ 9 ]. Notably, the body weights of Apoe −/− Nod1 −/− and Apoe −/− mice remained similar after 16 weeks with HFD (Figure S1a). Likewise, the plasma lipid concentrations after the HFD regimen did not show statistical differences when comparing Apoe−/−Nod1−/−and Apoe−/−groups (Figure S1b). When we evaluated the expression of SMA, NOD1, and MAC3 in consecutive sections of human atherosclerotic coronary arteries, we observed a marked NOD1 staining in cells near the lipid deposition areas, while the staining was much weaker in the non-atherosclerotic coronary controls. In fact, immunohistochemical analysis of human plaques of SMC and macrophages markers revealed elevated expression of NOD1 in both cell types (Figure 1c and Figure S2). These results, together with our previous studies [ 9 ] suggest a critical role for NOD1 in the key cell players of human and mice atherosclerosis. The accumulation of oxLDLs in the intima is crucial in the plaque lifetime. Given the fact that these oxidized lipids tightly regulate Nod1 expression, we next determined whether they could also regulate its expression in the two main cell components of the plaque. Treatment of BMDM and SMC with oxLDL, but not with LDL (used as controls for lipid composition of the culture medium), activated the NOD1 signaling cascade in both cell types, an effect suppressed after NOD1 inhibition by Nodinitib-1 (Figure 1d and Figure S3 for quantification of the blots). Moreover, NOD1 activation with the agonist iE-DAP, but not the inactive analogue iE-Lys, promoted RIP-2 and p65 phosphorylation, an effect suppressed when macrophages were treated with Nodinitib-1 (Figure 1d) . The effects on SMC were less intense, but inhibition of NOD1 by Nodinitib-1 confirmed the relevance of this innate Cells 2020,9, 2067 6 of 14 immune receptor in RIP-2 signaling in these cells (Figure 1d). SMC proliferation and macrophage foam cell formation are especially important hallmarks in advanced atherosclerosis lesions [ 4 , 19 ]. Therefore, we analyzed in vitro foam cell development in both cell types in response to oxLDL. ORO staining of intracellular lipids indicated that while macrophages and SMC isolated from Apoe −/− Nod1 −/− and Apoe −/− mice engulfed similarly unmodified LDL, fewer Apoe −/− Nod1 −/− macrophages and SMC engulfed oxLDL (Figure 1e). Analysis of the scavenger receptor CD36, Olr1 (Lox1, oxidized low-density lipoprotein receptor 1) and Cd68 (LDL scavenger receptor) mRNA in the aortic arch of HFD fed Apoe −/− and Apoe −/− Nod1 −/− did not show differences between both groups; however, a modest increase in Msr1 (Sr-a; macrophage scavenger receptor 1) mRNA, but statistically significant, was measured, indicating that lipid uptake via these receptors was not reduced by Nod1 deficiency [ 20 – 22 ] (Figure S4). The analysis of total lipid in the aortic valve of the same mice (Figure S5) did not reveal a biological significance for NOD1 in foam-cell formation in advanced atherosclerosis. 3.2. NOD1 Deficiency Modulates Structural and Compositional Features of Vulnerable Plaques We also examined the consequences of inactivating Nod1 on plaque composition. Human vulnerable plaques are typically associated with the presence of a highly inflammatory cell content and a large NC covered by a thin FC, the latter being characterized by decreased content of SMC and collagen [ 23 ]. The extent of the NC in relation to the size of the plaque was greater in Apoe −/− than in Apoe −/− Nod1 −/− mice, while the FC was thicker in Apoe −/− Nod1 −/− mice (Figure 2a). Consistent with these results, collagen evaluation by picosirius red staining showed a higher percentage of total positive area within the intima in Apoe −/− Nod1 −/− than in Apoe −/− mice. Further analysis of collagen composition revealed a significant difference between type I and type III collagen, demonstrating a higher proportion of thick fibers in Apoe −/− Nod1 −/− mice than in Apoe −/− mice (Figure 2a, inset). Therefore, although Apoe −/− mice are not particularly prone to develop unstable plaques [ 24 ], our analysis points towards the idea that Nod1 deficiency might favor plaque-stabilizing factors. To further assess the progression of atheromas in Apoe −/− Nod1 −/− and Apoe −/− mice, we determined the inflammatory and the SMC content in the aortic cusps, the region containing the most advanced lesions in the Apoe-deficient model [ 25 ]. Analysis of the aortic root revealed a lower content of intimal neutrophils and macrophages in Apoe −/− Nod1 −/− lesions compared to Apoe −/− mice. Not only this, but the intimal positive area for smooth-muscle actin was significantly higher in Apoe −/− Nod1 −/− than in Apoe −/− mice, compatible with a lower overall vulnerability in Apoe −/− Nod1 −/− atherosclerotic lesions (Figure 2b). These results are consistent with those obtained when analyzing the leukocyte content in the blood and in the spleen (Figures S6 and S7). 3.3. Nod1 Inactivation Increases SMC Proliferation and Reduces Macrophage Apoptosis We next seek to investigate the mechanisms underlying the atheroprotective action of the Nod1 deletion in both SMC and macrophages. Cell proliferation and apoptosis play equally important roles in atherosclerosis [ 26 ]. Interestingly, Apoe −/− Nod1 −/− SMC entered earlier into the S phase of the cell cycle compared to Apoe −/− controls, as determined by flow cytometry analysis of starvation-synchronized cultures (Figure S8). These studies demonstrated a significantly higher percentage of cells in G 1 /G 0 and S-phases in Apoe −/− vs. Apoe −/− Nod1 −/− SMC at all-time points analyzed (0, 12, and 24 h). Moreover, the differences in the percentages of Apoe −/− cell cultures in S-phase reached statistical significance after 24 h of serum restimulation compared to 0 h (p-value <0.01), while in Apoe −/− Nod1 −/− , differences in S-phase reached statistical significance 12 h after serum restimulation (p-value <0.05). Taken together, these findings suggest that serum-restimulated Apoe −/− Nod1 −/− SMC re-enter the cell cycle faster than Apoe −/− controls. Despite the increased proliferative capacity of cultured SMC lacking Nod1, cell proliferation was similar in cross-sections of the aortic root from Apoe −/− Nod1 −/− and Apoe −/− mice fed HFD for 16 weeks (Figure 3a), as estimated by the neointimal content of vascular smooth muscle and macrophages positives for Ki67. Cells 2020,9, 2067 7 of 14 Cells 2020, 9, x FOR PEER REVIEW 6 of 15 Figure 1. Activation of nucleotide-binding oligomerization domain (NOD)-1 signaling pathway in macrophages and smooth muscle cells (SMC) in advanced atherosclerosis. (a) Quantification of lesion area in the semilunar valve cusps of Apoe−/− (n = 14) and Apoe−/−Nod1−/− (n = 12) mice fed high-fat diet (HFD) for 16 weeks. (b) Quantification of positive Oil Red O (ORO) lesion area in the indicated aortic regions in the same cohort. Panels show representative en face ORO staining of aortas from these animals. (c) Representative images for co-localization of smooth muscle α-actin (SMA), NOD1 and Mac3 immunohistochemistry in the arterial intimal thickening of non-atherosclerotic (non-athero.) and atherosclerotic (athero.) human coronary arteries. Arrows point out NOD1+ cells of macrophages (blue) and smooth muscle cells (black) in the lesion area. Red arrows delimit internal elastic lamina. (d) Immunoblot analysis and representative panel of NOD1, pRIP2, RIP2, pP65, and P65 in Wt BMDM and SMC pre-treated with the NOD1 inhibitor Nodinitib-1 and/or stimulated with native LDL (as control for lipid load in the medium), oxLDL, iE-Lys (an inactive NOD1 activator) and c12-iE-DAP (an agonist for NOD1) for 24 h or 48 h, respectively. Protein levels were normalized to tubulin. (e) Apoe−/− and Apoe−/−Nod1−/− macrophages (Mφ) and SMC were exposed to native LDL or oxLDL for 24 h or 48 h respectively, and then ORO stained. Representative images for oxLDL treatment and Figure 1. Activation of nucleotide-binding oligomerization domain (NOD)-1 signaling pathway in macrophages and smooth muscle cells (SMC) in advanced atherosclerosis. ( a ) Quantification of lesion area in the semilunar valve cusps of Apoe −/− (n=14) and Apoe −/− Nod1 −/− (n=12) mice fed high-fat diet (HFD) for 16 weeks. ( b ) Quantification of positive Oil Red O (ORO) lesion area in the indicated aortic regions in the same cohort. Panels show representative en face ORO staining of aortas from these animals. ( c ) Representative images for co-localization of smooth muscle α -actin (SMA), NOD1 and Mac3 immunohistochemistry in the arterial intimal thickening of non-atherosclerotic (non-athero.) and atherosclerotic (athero.) human coronary arteries. Arrows point out NOD1 + cells of macrophages (blue) and smooth muscle cells (black) in the lesion area. Red arrows delimit internal elastic lamina. ( d ) Immunoblot analysis and representative panel of NOD1, pRIP2, RIP2, pP65, and P65 in Wt BMDM and SMC pre-treated with the NOD1 inhibitor Nodinitib-1 and/or stimulated with native LDL (as control for lipid load in the medium), oxLDL, iE-Lys (an inactive NOD1 activator) and c12-iE-DAP (an agonist for NOD1) for 24 h or 48 h, respectively. Protein levels were normalized to tubulin. ( e )Apoe −/− and Apoe −/− Nod1 −/− macrophages (M ϕ ) and SMC were exposed to native LDL or oxLDL for 24 h or 48 h respectively, and then ORO stained. Representative images for oxLDL treatment and quantification are shown of stained cells in three independent experiments. Data are represented as mean ± s.e.m. of the indicated number (n) of repeats. * p<0.05, *** p<0.001 **** p<0.0001 vs. Apoe −/− by Student’s ttest. Bars, 100 µm (a), 1 mm (b), 50 µm (c,e). Cells 2020,9, 2067 8 of 14 Cells 2020, 9, x FOR PEER REVIEW 8 of 15 Figure 2. Nod1 deficiency in Apoe−/− mice results in decreased vulnerable plaques. (a) The collagen content of aortic sinus plaques in Apoe−/− (n = 13) and Apoe−/−Nod1−/− (n = 11) mice fed HFD for 16 weeks was evaluated by Sirius red staining. Quantification of total collagen content, necrotic core (NC) area and fibrous cap (FC) thickness was analyzed using brightfield microscopy. Hughe birefringence under polarized light illumination allowed quantification of type I (‘mature-collagen’; orange red birefringence) or type III (‘immature-collagen’; green yellow birefringence and green image in the inset) collagen as percent of total collagen. Representative images in bright field and polarized light illumination in lesions of the aortic sinus are shown. (b) Quantification of macrophage, neutrophil and SMC neointimal content in the semilunar valve cusps of the same cohort of mice. Vulnerability plaque index was determined as the NC and MAC3+ areas divided by the collagen and smooth muscle α-actin (α-SMA)+ staining areas of the same lesions [24]. Representative immunofluorescence images of anti-MAC3, anti-Ly6G and anti-α-SMA staining in lesions of the aortic sinus are shown. Data are represented as mean ± s.e.m. of the indicated number (n) of repeats. **p < 0.01, ***p < 0.001 vs. Apoe−/− by Student’s t test. Bars, 50 µm. To further assess the progression of atheromas in Apoe−/−Nod1−/− and Apoe−/− mice, we determined the inflammatory and the SMC content in the aortic cusps, the region containing the most advanced lesions in the Apoe-deficient model [25]. Analysis of the aortic root revealed a lower content of intimal Figure 2. Nod1 deficiency in Apoe −/− mice results in decreased vulnerable plaques. ( a ) The collagen content of aortic sinus plaques in Apoe −/− (n=13) and Apoe −/− Nod1 −/− (n=11) mice fed HFD for 16 weeks was evaluated by Sirius red staining. Quantification of total collagen content, necrotic core (NC) area and fibrous cap (FC) thickness was analyzed using brightfield microscopy. Hughe birefringence under polarized light illumination allowed quantification of type I (‘mature-collagen’; orange red birefringence) or type III (‘immature-collagen’; green yellow birefringence and green image in the inset) collagen as percent of total collagen. Representative images in bright field and polarized light illumination in lesions of the aortic sinus are shown. ( b ) Quantification of macrophage, neutrophil and SMC neointimal content in the semilunar valve cusps of the same cohort of mice. Vulnerability plaque index was determined as the NC and MAC3 + areas divided by the collagen and smooth muscle α -actin ( α -SMA)+staining areas of the same lesions [ 24 ]. Representative immunofluorescence images of anti-MAC3, anti-Ly6G and antiα -SMA staining in lesions of the aortic sinus are shown. Data are represented as mean ± s.e.m. of the indicated number (n) of repeats. ** p<0.01, *** p<0.001 vs. Apoe −/− by Student’s ttest. Bars, 50 µm. Cells 2020,9, 2067 9 of 14 Cells 2020, 9, x FOR PEER REVIEW 10 of 15 Figure 3. Lack of Nod1 reduces apoptotic activity of SMC and macrophages. (a) Cross-sections from the aortic sinus of Apoe−/− (n = 6) and Apoe−/−Nod1−/− (n = 6) mice fed HFD for 16 weeks were doubly stained with Ki-67/MAC-3, cleaved caspase-3/MAC-3 (top) or Ki-67/smooth muscle α-actin (α-SMA), cleaved caspase-3/α-SMA (bottom) to appropriately identify not only proliferative macrophages or in apoptosis (top), but also SMC (bottom) in proliferative or apoptotic state. The results for proliferation are presented as percentage of cells doubly positive for Ki-67 and MAC-3 or α-SMA relative to total number of Ki-67+ cells within the atheroma. The results for apoptosis are presented as percentage of cells doubly positive for cleaved caspase-3 and MAC-3 or α-SMA relative to total number of cleavedcaspase-3+ cells within the atheroma. SMC (b) and macrophage (Mφ) (c) apoptotic cells were identified as the sub-G0 population after propidium iodide staining by flow cytometry (upper panels) or as cleaved caspase-3 immunoreactive cells by confocal microscopy (lower panels). Cells were either untreated, irradiated with ultraviolet (UV) light (80 J/m2 and harvested after 24 h for BMDM; 120 J/m2 and harvested after 48 h for SMC), or incubated with 1 mM S-nitrosoglutathione (GSNO), 50 µg/mL oxidized LDL (oxLDL) or 1 µg/mL c12-iE-DAP for 24 h (BMDM) or 48 h (SMC). Results using both Figure 3. Lack of Nod1 reduces apoptotic activity of SMC and macrophages. ( a ) Cross-sections from the aortic sinus of Apoe −/− (n=6) and Apoe −/− Nod1 −/− (n=6) mice fed HFD for 16 weeks were doubly stained with Ki-67/MAC-3, cleaved caspase-3/MAC-3 (top) or Ki-67/smooth muscle α -actin ( α -SMA), cleaved caspase-3/ α -SMA (bottom) to appropriately identify not only proliferative macrophages or in apoptosis (top), but also SMC (bottom) in proliferative or apoptotic state. The results for proliferation are presented as percentage of cells doubly positive for Ki-67 and MAC-3 or α -SMA relative to total number of Ki-67 + cells within the atheroma. The results for apoptosis are presented as percentage of cells doubly positive for cleaved caspase-3 and MAC-3 or α -SMA relative to total number of cleaved-caspase-3 + cells within the atheroma. SMC ( b ) and macrophage (M ϕ ) ( c ) apoptotic cells were identified as the sub-G0 population after propidium iodide staining by flow cytometry (upper panels) or as cleaved caspase-3 immunoreactive cells by confocal microscopy (lower panels). Cells were either untreated, irradiated with ultraviolet (UV) light (80 J/m 2 and harvested after 24 h for BMDM; 120 J/m 2 and harvested after 48 h for SMC), or incubated with 1 mM S-nitrosoglutathione (GSNO), 50 µ g/mL oxidized LDL (oxLDL) or 1 µ g/mL c12-iE-DAP for 24 h (BMDM) or 48 h (SMC). Results using both methods represent the average of three independent experiments. Data are represented as mean ± s.e.m. of the indicated number (n) of repeats. *p<0.05, **p<0.01, ****p<0.0001 vs. Apoe −/− by Mann–Whitney U test ( a ) or by Student’s ttest (b,c). Bars, 20 µm.