Cardiotrophin-1 Deficiency Abrogates Atherosclerosis Progression

Cardiotrophin-1 (CT-1) is associated with cardiovascular (CV) diseases. We investigated the effect of CT-1 deficiency in the development and progression of atherosclerosis in double knockout Apoe−/−ct-1−/− mice. Apoe−/− C57Bl/6 or Apoe−/−ct-1−/− C57Bl/6 mice were fed a normal chow diet (NCD) or a high-cholesterol diet (HCD). After sacrifice, serum triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), free fatty acids and systemic paracrine factors were measured. Intraplaque lipid and collagen content were quantified in the aortic sections. Immune cell populations in spleen, lymph nodes and aorta were analysis by flow cytometry. Apoe−/−ct-1−/− mice in accelerated atherosclerosis exhibited a reduction of total cholesterol, LDL-C, atherosclerotic plaques size in the aortic root and in the abdominal aorta and improved plaque stability in comparison to Apoe−/− mice. CT-1 deficiency in Apoe−/− mice on (HCD) promoted atheroprotective immune cell responses, as demonstrated by a rise in plasma anti-inflammatory immune cell populations (regulatory T cells, Tregs; regulatory B cells, Bregs and B1a cells) and atheroprotective IgM antibodies. CT-1 deficiency in advanced atherosclerosis mediated regulation of paracrine factors, such as interleukin (IL)-3, IL-6, IL-9, IL-15, IL-27, CXCL5, MCP-3, MIP-1α and MIP-1β. In a model of advanced atherosclerosis, CT-1 deficiency induced anti-inflammatory and atheroprotective effects which resulted in abrogation of atheroprogression.

Cardiovascular (CV) diseases are the leading cause of death and morbidity in developed countries 1 . The underlying cause of the most serious CV events is atherosclerosis, which is defined as a chromic inflammatory disease characterized by the build-up of subendothelial cholesterol deposits and the formation of leukocyte-rich plaques in the intimal layer of arteries. The fibrous cap is an atheroprotective layer of vascular smooth muscle cells (VSMCs) that covers the atherosclerotic plaque 2 and induces acute thrombo-occlusive events, such as myocardial infarction and stroke 3 . Immune cells and inflammation play a key role in promoting the disruption of the fibrous cap 2 . Full comprehension of the mechanisms of atherosclerosis is linked to revealing the role of the paracrine mediators released by the heterogenous cell populations involved in the development, progression, and complications of atherosclerosis.
Cardiotrophin-1 (CT-1) is a member of the interleukin (IL)-6 family of cytokines, and was initially cloned based on its ability to induce hypertrophy in neonatal cardiomyocytes 4 . CT-1 is highly expressed in the cells of the cardiovascular system -cardiomyocytes, cardiac fibroblasts, vascular endothelial cells, vascular smooth muscle cells (VSMCs), macrophages [5][6][7][8] , as well as in other organs 9,10 . Factors like mechanical stretching, hypoxia, angiotensin II, aldosterone, growth factors, insulin, glucose, reactive oxygen species [11][12][13][14][15][16] , hypertension 17 and pressure overload 18 trigger CT-1 expression. CT-1 binds to glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR) 4 and induces cardiac protection via inhibition of apoptosis, suggesting a protective role of CT-1 in response to acute hypoxia in vivo 19 which is in line with other studies showing the anti-apoptotic effect of CT-1 treatment on embryonic, neonatal, and adult cardiomyocytes 6,20,21 . On the other hand CT-1 has been shown promotes cardiac hypertrophy 22 , atherosclerosis, arterial stiffness 23 and vascular inflammation 5 . Elevated CT-1 CT-1 deficiency reduces atherosclerosis lesion size and modulates parameters of plaque stability in Apoe −/− mice on high cholesterol diet. To determine the pathological function of CT-1 in atherogenesis, we examined whether CT-1 deletion alters atherosclerotic plaque size and stability. Apoe −/− mice and Apoe −/− ct-1 −/− mice were fed NCD for 16 weeks or HCD for 11 weeks. Atherosclerotic lesion size in the aortic roots was significantly increased in Apoe −/− mice on HCD versus Apoe −/− mice on NCD (Fig. 2a,b), and as also evident in the representative picture of Oil Red O staining (Fig. 2c). In contrast, Apoe −/− ct-1 −/− mice on HCD exhibited a 1.8-fold reduction in lesion size area in the aortic root ( Fig. 2a) versus Apoe −/− mice on HCD. Atherosclerotic lesion size, expressed as a percentage in the aortic roots and abdominal part of the aorta, was significantly increased in Apoe −/− mice on HCD versus Apoe −/− mice on NCD (Fig. 2b,d). During spontaneous atherosclerosis, Apoe −/− ct-1 −/− mice showed a 1.2-fold increase in lesion size compared to Apoe −/− mice on NCD, expressed as the percentage of total lesion area in the aortic root (Fig. 2b). However, in accelerated atherosclerosis (HCD), lesion size reduction in Apoe −/− ct-1 −/− versus Apoe −/− mice was 1.2-fold in the aortic root ( Fig. 2b) and 3.2-fold in the abdominal aorta (Fig. 2d), as illustrated in the representative images of Oil Red O staining (Fig. 2c,e). HCD induced significant increase in the percentage of lesion CD68 macrophages (Fig. 3a,b) and neutrophils Ly6G cells (Fig. 3c,d) in the aortic roots of Apoe −/− mice, while CT-1 deficiency had not effect on macrophages or neutrophils accumulation in the aortic roots under NCD or HCD. Collagen plays a key role in determining plaque stability and protects plaque against rupture 31 . Picrosirius red staining of the atherosclerotic lesions in the aortic roots revealed a significant reduction of collagen accumulation in Apoe −/− ct-1 −/− versus Apoe −/− mice on NCD (Fig. 4a,b). However, in accelerated atherosclerosis the collagen content was increased by 1.6-fold in Apoe −/− ct-1 −/− versus Apoe −/− mice on HCD (Fig. 4a,b). In parallel, CT-1 deficiency in Apoe −/− on HCD resulted in a significant reduction in the necrotic core area (Fig. 4c,d) and an increase in the fibrous core thickness in Apoe −/− ct-1 −/− mice on both NCD and HCD (Fig. 4e,f) versus Apoe −/− mice. Importantly, the increase in the collagen content in Apoe −/− ct-1 −/− mice on HCD was associated with a prominent increase of 2-fold α-SMA expression in the atherosclerotic plaques of Apoe −/− ct-1 −/− mice (Fig. 4g,h) versus Apoe −/− mice on HCD. In addition, Apoe −/− ct-1 −/− exhibited on HCD a 1.9-fold reduction in MMP9 expression in the atherosclerotic roots versus Apoe −/− mice (Fig. 4i,j). Interestingly, the levels of pro-MMP-9 in the serum of Apoe −/− mice on HCD were reduced versus Apoe −/− mice on NCD, while Apoe −/− ct-1 −/− mice on HCD had significantly higher pro-MMP9 levels (Fig. 4k), indicating a possible reduction in the proteolysis of the pro-peptide to active MMP9 in the serum of Apoe −/− ct-1 −/− mice on HCD. The present findings indicate that CT-1 deficiency in www.nature.com/scientificreports www.nature.com/scientificreports/ Apoe −/− mice in accelerated atherosclerosis not only results in reduced atherosclerotic lesion size, but it also modulates key aspects of the plaque structure stability with major clinical consequences.

Discussion
The present study provides compelling evidence of the critical function of CT-1 in the pathogenesis of atherosclerosis. In accelerated atherosclerosis, CT-1 deficiency appears to induce a multitude of beneficial effects. Significantly, CT-1 abrogation not only resulted in lower cholesterol levels and smaller atherosclerotic plaques, but it also promoted systemic immunomodulatory effects, including 1) induction of atheroprotective immune cell populations -Tregs, Bregs and B1a cells; 2) regulation of systemic inflammatory factors; and 3) increased levels of circulating IgM, the concentration of which is known to be inversely correlated with atherosclerosis 32 . Moreover, Apoe −/− ct-1 −/− mice exhibited a number of changes in the fibrous cap formation known to be beneficial for plaque stability in atherosclerosis, in particular higher collagen accumulation, preserved α-SMA expression, and smaller necrotic cores, thicker fibrous caps and lower MMP9 expression in the aortic roots. Taken together, these observations have important implications. First, CT-1 undoubtedly emerges as a key factor in the pathogenesis of atherosclerosis; and second, CT-1 inhibition results in abrogation of atherosclerotic disease progression.
One of the most intriguing findings of the present study is the effect of CT-1 deficiency on the cholesterol levels and atherosclerotic plaque size in Apoe −/− mice in accelerated atherosclerosis. Plasma total cholesterol and LDL levels are highly correlated with the extent of coronary atherosclerosis 33 , while hypercholesterolemia also enhances local and systemic proinflammatory responses 34 . Therefore, the reduction of LDL observed in Apoe −/− ct-1 −/− mice on HCD has two very significant consequences: on the one hand, it directly results in diminished LDL retention in the subendothelial space, leading to less atherosclerotic plaque formation; on the other hand, it indirectly leads to reduced inflammation. As a result, Apoe −/− ct-1 −/− mice on HCD exhibited atherosclerotic plaque sizes comparable to Apoe −/− mice on NCD, however no difference in macrophages nor neutrophils accumulation in the roots was observed. Particularly impressive in accelerated atherosclerosis was the reduction in the size of the atherosclerotic lesions in the abdominal aorta of Apoe −/− ct-1 −/− mice. However, Apoe −/− ct-1 −/− mice develop bigger atherosclerotic lesions expressed as a percentage of roots area during spontaneous atherosclerosis, which could be probably associated with the anti-apoptotic effect mediated by CT-1 6,20,21 and in response to acute hypoxia in vivo 19 . During early atherosclerosis, the reduction of apoptotic cells accumulation has been shown to limit local inflammation and lesion growth by preventing secondary cellular necrosis 35 , the increased lesion size in CT-1 deficient Apoe −/− mice during spontaneous atherosclerosis could be linked to the abrogation of the anti-apoptotic effect of CT-1. However, our findings highlight, that deficiency of CT-1 becomes particularly beneficial in accelerated atherosclerosis where plaque complexity increases, and inflammatory and immune processes start to play an important role in plaque progression.
The majority of coronary thrombosis events are caused by plaque rupture 36 . The structure, composition, and turnover of the extracellular matrix (ECM) are crucial for atherosclerotic plaque stability. Picrosirius red staining of atherosclerotic lesions in the aortic sinus revealed a significant increase in collagen content in Apoe −/− ct-1 −/− mice with accelerated atherosclerosis, in parallel with a prominent reduction of MMP9 expression and necrotic core and increase in fibrous cap thickness and α-SMA expression in the atherosclerotic roots. A well-established dynamic equilibrium of ECM production and degradation is an essential prerequisite for determining plaque stability. In this regard, it is well known that collagen accumulation prevents plaque rupture 31 , while matrix-degrading proteases like MMP-9 degrade components of the extracellular matrix. MMP-9 is a protein expressed and secreted in an inactive form named pro-MMP-9, which is then activated by proteolysis of the propeptide domain 37 . MMP-9 is abundantly expressed in atherosclerotic plaques with a vulnerable histological appearance 38 . Interestingly, the levels of pro-MMP-9 in the serum of Apoe −/− mice on HCD were tendentially reduced, while Apoe −/− ct-1 −/− mice on HCD showed upregulated pro-MMP9 levels, indicating a reduction in proteolysis of the propeptide to active MMP9 in the serum of Apoe −/− ct-1 −/− mice in accelerated atherosclerosis. CT-1 deficiency of Apoe −/− mice in accelerated atherosclerosis not only diminished atherosclerotic lesion size, but it also improves the plaque structure stability parameters known to have a critical clinical importance due to atherosclerotic plaque ruptures.
Atherosclerosis is characterized by a chronic, low-grade inflammatory response associated with infiltration of immune cells into the atherosclerotic plaque 39 . Moreover, LDL acts as a self-antigen that drives an autoimmune response against self-proteins in the atherosclerotic plaque 40 . Importantly, CT-1 abrogation promoted Tregs cells, shown to mediate protective effects in atherosclerosis 41 through the release of anti-inflammatory cytokines and suppression of autoreactive effector T cells. n = 6-8/group and ** p < 0.01 expressed as % of aortic root area. (i) Bar graphs represent the mean ± SEM of MMP9 expressed as % of aortic root area in Apoe −/− or Apoe −/− ct-1 −/− mice on NCD or HCD, as indicated, and (j) representative pictures of MMP9 in atherosclerotic roots in Apoe −/− or Apoe −/− ct-1 −/− mice on NCD or HCD, as indicated, with n = 6-8/group and *p < 0.05, and ***p < 0.001 expressed as % of aortic root area. (k) Bar graphs represent the mean ± SEM of pro-MMP9 quantification in the serum of Apoe −/− or Apoe −/− ct-1 −/− mice on NCD or HCD, as indicated, with n = 6-8/group and *p < 0.05. The scale bar is 200 μm. www.nature.com/scientificreports www.nature.com/scientificreports/ Moreover, CT-1 deficiency in Apoe −/− mice has a profound effect on Th1, Th2 and Th17 cells under HCD and Th17 cells also under NCD. While Th1 cells are known to be highly inflammatory and to promote and accelerate lesion development 42 , the role of Th2 and Th17 cells is less clear, however CT-1 deficiency seems to greatly reduce www.nature.com/scientificreports www.nature.com/scientificreports/ the percentage of Th1, Th2 and Th17 systemically which could result in regulation in the systemic inflammation. In parallel, Bregs is known to exert vascular protective functions via the release of soluble factors, such as IL-10, TGF-β and IL-35 43 , and levels were increased in the SP of Apoe −/− ct-1 −/− mice on HCD. Conversely, it is known that increased levels of TGFβ1 correlate with accelerated atherosclerosis 44 and induction of vascular calcification 45 . Importantly, Bregs producing TGF-β in the PLN and SP of Apoe −/− ct-1 −/− mice on HCD were reduced. This important observation indicates that the induction of atheroprotective Bregs does not lead to increased atherosclerosis, promoting TGF-β production. In accelerated atherosclerosis, Apoe −/− ct-1 −/− mice exhibited an expansion of the atheroprotective B1a cells in the SP, as well as elevated IgM 46,47 . B1a cells are known to secrete natural IgM, which is deposited in atherosclerotic lesions where it mediates necrotic core reduction 47 . However, it has been shown that the level of circulating oxLDL IgM autoAbs is lower in CAD patients than in no CAD patients, supporting the hypothesis that the oxLDL IgM autoAbs might be inversely associated with the presence of atherosclerosis 48 , while anti-oxLDL, anti-oxLDL-β2GPI IgM and anti-oxLDL-β2GPI IgG may increase the risk for cardiovascular diseases in systemic lupus erythematosus patients 49 . CT-1 deficiency promoted a significant reduction in the levels of IgM to oxLDL in Apoe −/− mice, which is in line with the discussed below findings, that oxLDL IgM autoAbs might be inversely associated with the extent of atherosclerosis. However, the role of circulating oxLDL IgM remains controversial, since it has also been demonstrated that IgM to oxLDL could play a protective role too, however in that study high-risk individuals were not selected, but subjects from a random population-based cohort were used and the underlying mechanisms were not examined either and the conclusion that IgM to oxLDL has a protective role was regarded as speculative 32 .
FOB cells are shown to promote atherosclerosis via antigen presentation to T FH cells and this interaction further promotes humoral responses by stimulating B cells 50 . In contrast, MZB cells limit an exaggerated adaptive immune response via T FH cell regulation 51 . In spite of the fact that CT-1 deficiency in accelerated atherosclerosis induced FOB cell production while at the same time promoting MZB cell reduction in the SP, www.nature.com/scientificreports www.nature.com/scientificreports/ Apoe −/− ct-1 −/− mice on HCD did not exhibit an increase in T FH cells, but showed a pronounced increase in Tregs and Bregs, and reduced atherosclerotic lesion size. Taken together, these findings indicate that inhibition of CT-1 expression in atherosclerosis promotes direct anti-inflammatory effects associated with an increase of atheroprotective immune cell populations.
Chronic inflammation driven by immune cells and cytokines plays a crucial role in the development and progression of atherosclerosis. CT-1 could enhance atherogenesis via induction of inflammatory and proatherogenic molecule expression 8,[26][27][28] . Consistently with these findings, inhibition of CT-1 expression in Apoe −/− mice in accelerated atherosclerosis resulted in inducing substantial changes in the systemic levels of several cytokines and chemokines. Like many other cytokines, IL-3 and IL-6 have both proinflammatory and anti-inflammatory properties. CT-1 deficiency in Apoe −/− mice on HCD resulted in elevated IL-3 systemic levels. IL-3 is secreted by activated T cells and stimulates multiple hematopoietic cell types involved in the immune response 52 , while IL-3 treatment in mice prevents mAb/LPS-induced arthritis by inhibiting inflammation 53 . Although IL-6 is a marker for systemic activation of proinflammatory cytokines 54 , it can act in an atheroprotective manner by increasing cholesterol efflux to apolipoprotein A1 (apoA1) in macrophages 55 . Importantly, Apoe −/− ct-1 −/− mice on HCD exhibited about a 2-fold increase in circulating levels of IL-6, but also a prominent reduction of cholesterol levels. Although IL-9, which is prominently elevated in Apoe −/− ct-1 −/− mice fed HCD, could be linked to the increase in Tregs in the SP of Apoe −/− ct-1 −/− mice fed HCD, as IL-9 is shown to protect Tregs against apoptosis and enhance their function 56   elevated in Apoe −/− ct-1 −/− mice fed HCD, and it could be linked with the atheroprotective role that IL-27 plays by regulating macrophage activation 58 . CT-1 deficiency in accelerated atherosclerosis promotes several cytokines with atheroprotective activities, like inflammation inhibition, plaque stabilization and control of macrophage activation, the net effect of which could partly explain anti-inflammatory effect observed after CT-1 abrogation in Apoe −/− ct-1 −/− mice fed HCD.
Chemokines not only direct leukocytes to sites of inflammation during atherogenesis, they also play a role in cell homeostasis and foam cell formation. For example, CXCL5, which was remarkably elevated in Apoe −/− ct-1 −/− mice fed HCD, has been shown to modulate macrophage activation, increase expression of the cholesterol efflux regulatory protein ABCA1, and enhance cholesterol efflux activity in macrophages 59 . Therefore, the increase in CXCL5 levels in Apoe −/− ct-1 −/− mice could be considered as an additional mechanism contributing to the reduction of total cholesterol as well as LDL-C. MCP-3 was shown to promote VSMCs proliferation 60 and increase plasma total cholesterol, atherosclerotic lesions, and hepatic lipid accumulation under atherogenic conditions 61 . In agreement with these findings, we observed an increase in MCP-3 levels in the circulation upon feeding Apoe −/− mice HCD, however, MCP-3 was significantly reduced in Apoe −/− ct-1 −/− mice fed HCD. MI1α and MI1β levels were elevated in Apoe −/− ct-1 −/− mice in accelerated atherosclerosis. MI1α (CCL3) deficiency in Ldlr −/− mice had no effect on atherosclerotic lesion formation in the aortic sinus 62 . The results demonstrate that CT-1 deficiency in accelerated atherosclerosis promotes the induction of anti-inflammatory mediators. Taken together, these results could partly explain the beneficial effect of CT-1 deficiency in atherosclerosis progression and development. CT-1 expression in the heart and liver, in immune and endothelial cells, and in VSMCs in the aorta of ageing Apoe −/− mice further highlights the involvement of CT-1 in the pathogenesis of atherosclerosis.
The present study undoubtedly demonstrates that CT-1 abrogation in accelerated atherosclerosis prevents atherosclerosis progression and development. A crucial question remains whether the presented atheroprotective effects of CT-1 deficiency in mice might be achieved in patients. Armed with a better understanding of the multiple atheroprotective effects mediated by CT-1 abrogation in accelerated atherosclerosis, we believe that CT-1 could be an efficient therapeutic target in patients with coronary atherosclerosis. Immunohistochemistry. Mouse aortic sinus was serially cut in 5 μm transversal sections, as previously described 66,67 . Sections from mouse specimens were fixed in acetone and immunostained with specific anti-mouse MMP-9 antibody (R&D Systems), anti-mouse α-SMA antibody (Thermo Fischer), anti-mouse Ly6G (BD Pharmingen) and anti-mouse CD68 (Serotec) staining in atherosclerotic roots. Quantification was performed using the MetaMorph or Definiens software. Results for other parameters were calculated as percentages of the stained area on total lesion area. www.nature.com/scientificreports www.nature.com/scientificreports/ Necrotic core and Fibrous cap thickness evaluation. To evaluation the necrotic core (NC), the criteria used by Thim et al. 68 were applied. NC was considered the areas within a lesion that were negative for Picrosirius red staining (i.e., extracellular matrix was absent in parallel with total or almost complete loss of collagen). Boundary lines were delineated around those regions and the area was measured by Fiji ImageJ image analysis softwareMeasurements were performed blindly to the study groups. NC percentage area was calculated by dividing total NC area by total lesion area. Fibrous cap thickness was assessed from the largest necrotic cores stained with Picrosirius red staining and measuring the thinnest section of the cap as determined by the distance between the outer edge of the cap and the NC border. Fibrous caps buried in deep necrosis areas were not considered. Measurements were performed blindly to the study groups.

Mice. Eleven-week old male Apoe
Oil Red O staining for lipid content. Five sections per mouse aortic roots and abdominal aorta were stained with Oil Red O, as previously described 66,67 . Sections and aortas were counter-stained with Mayer's hemalum solution and rinsed in distilled water. Quantification was performed using the MetaMorph software. Data were calculated as the percentage of the stained area from total lesion area.
Picrosirius red staining. Five sections per mouse aortic sinus were rinsed with water and incubated with 0.1% Sirius red (Sigma Chemical Co, St Louis, MO, USA) in saturated picric acid for 90 min. Sections were rinsed twice with 0.01 M HCl for 1 min and then immersed in water. After dehydration with ethanol for 30 seconds and cover-slipping, pictures of the sections were taken with ordinary polychromatic microscopy with identical exposure settings. Total collagen content was evaluated under polychromatic light 69,70 . Quantifications were performed with Definiens Developer 2.7 software (Definiens Inc). Data were calculated and presented as the percentage of the stained area on total lesion area.

Measurement of ox-LDL-specific IgM Abs.
The ox-LDL-specific Abs were measured using ELISA.
Briefly, copper-oxidized human LDL and native human LDL (Thermo Fisher) were used to coat 96-well ELISA plates at 50 μl of 10 μg/ml overnight at 4 °C. Duplicate samples of 50 μl mouse serum diluted 1:80 were added into the ELISA plates for 1 h at 37 °C after blocking with 2% BSA, 5% FBS, followed by addition of anti-mouse IgM Abs conjugated with HRP (BD Pharmingen). Color development was done by addition of TMB solution, and plates were read at 450 nm wavelength. ox-LDL-specific Ab was determined by subtracting the native LDL OD from the ox-LDL OD.
Protein extraction and western blot analysis. Liver, heart and SP of WT, ct-1 −/− , Apoe −/− and Apoe −/− ct-1 −/− mice were dissected, cut into pieces and lysed with lysis buffer containing CHAPS solution (Sigma) and complete mini inhibitors. Denaturized proteins were electrophoresed on NUPAGE 4-12% BT GEL 1.5 mm (Thermos Fisher), followed by transfer onto nitrocellulose membranes using a semi-dry transfer method (iBlot ™ 2 Transfer Stacks, Thermo Fisher) followed by blocking. Blots were incubated with primary antibodies against CT-1 antibody AF438 (1:1000; R&D) or β-Tubulin. The bound antibodies were detected by the respective horseradish peroxidase-conjugated secondary antibodies and visualized with SuperSignal ™ West Pico PLUS Chemiluminescent Substrate kit (Thermo Scientific). The resulted chemiluminescence was measured by using iBright Western Blot Imaging Systems (Thermo Scientific).
Statistical analysis. Statistics were performed using GraphPad Prism 8. For comparison of two groups of continuous variables, two-tailed unpaired Mann-Whitney U-tests with a confidence level of 95% were conducted if data were non-normally distributed. For multiple group comparison, two-tailed two-ways ANOVA was performed. The number of mice used for each analysis is indicated in the figure legends. All data are presented as the mean ± SEM and the statistical significance threshold used is p ≤ 0.05. *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005.