TL1A inhibits atherosclerosis in apoE-deficient mice by regulating the phenotype of vascular smooth muscle cells

TNF ligand-related molecule 1A (TL1A) is a vascular endothelial growth inhibitor to reduce neovascularization. Lack of apoE a expression results in hypercholesterolemia and atherosclerosis. In this study, we determined the precise effects of TL1A on the development of atherosclerosis and the underlying mechanisms in apoE-deficient mice. After 12 weeks of pro-atherogenic high-fat diet feeding and TL1A treatment, mouse aorta, serum, and liver samples were collected and used to assess atherosclerotic lesions, fatty liver, and expression of related molecules. We found that TL1A treatment significantly reduced lesions and enhanced plaque stability. Mechanistically, TL1A inhibited formation of foam cells derived from vascular smooth muscle cells (VSMCs) but not macrophages by activating expression of ABC transporter A1 (ABCA1), ABCG1, and cholesterol efflux in a liver X receptor–dependent manner. TL1A reduced the transformation of VSMCs from contractile phenotype into synthetic phenotypes by activating expression of contractile marker α smooth muscle actin and inhibiting expression of synthetic marker osteopontin, or osteoblast-like phenotype by reducing calcification. In addition, TL1A ameliorated high-fat diet–induced lipid metabolic disorders in the liver. Taken together, our work shows that TL1A can inhibit the development of atherosclerosis by regulating VSMC/foam cell formation and switch of VSMC phenotypes and suggests further investigation of its potential for atherosclerosis treatment. TNF ligand-related molecule 1A (TL1A) is a vascular endothelial growth inhibitor to reduce neovascularization. Lack of apoE a expression results in hypercholesterolemia and atherosclerosis. In this study, we determined the precise effects of TL1A on the development of atherosclerosis and the underlying mechanisms in apoE-deficient mice. After 12 weeks of pro-atherogenic high-fat diet feeding and TL1A treatment, mouse aorta, serum, and liver samples were collected and used to assess atherosclerotic lesions, fatty liver, and expression of related molecules. We found that TL1A treatment significantly reduced lesions and enhanced plaque stability. Mechanistically, TL1A inhibited formation of foam cells derived from vascular smooth muscle cells (VSMCs) but not macrophages by activating expression of ABC transporter A1 (ABCA1), ABCG1, and cholesterol efflux in a liver X receptor–dependent manner. TL1A reduced the transformation of VSMCs from contractile phenotype into synthetic phenotypes by activating expression of contractile marker α smooth muscle actin and inhibiting expression of synthetic marker osteopontin, or osteoblast-like phenotype by reducing calcification. In addition, TL1A ameliorated high-fat diet–induced lipid metabolic disorders in the liver. Taken together, our work shows that TL1A can inhibit the development of atherosclerosis by regulating VSMC/foam cell formation and switch of VSMC phenotypes and suggests further investigation of its potential for atherosclerosis treatment. Atherosclerosis is one of the common causes of cardiovascular diseases, which can seriously impair human health. It is also a complex pathophysiological process, characterized by disorders of cholesterol/lipid metabolism, inflammation, and ultimately the development of fibrous plaques in the aorta (1Wolf D. Ley K. Immunity and inflammation in atherosclerosis.Circ. Res. 2019; 124 (30653442): 315-32710.1161/CIRCRESAHA.118.313591Crossref PubMed Scopus (411) Google Scholar, 2Weber C. Noels H. Atherosclerosis: current pathogenesis and therapeutic options.Nat. Med. 2011; 17 (22064431): 1410-142210.1038/nm.2538Crossref PubMed Scopus (1460) Google Scholar). Previous studies have demonstrated that monocyte/macrophage accumulation and infiltration, endothelial cell dysfunctions, and vascular smooth muscle cell (VSMC) phenotypic differentiation play important roles in the development of atherosclerosis (3Gimbrone Jr., M.A. García-Cardeña G. Endothelial cell dysfunction and the pathobiology of atherosclerosis.Circ. Res. 2016; 118 (26892962): 620-63610.1161/CIRCRESAHA.115.306301Crossref PubMed Scopus (1342) Google Scholar, 4Bennett M.R. Sinha S. Owens G.K. Vascular smooth muscle cells in atherosclerosis.Circ. Res. 2016; 118 (26892967): 692-70210.1161/CIRCRESAHA.115.306361Crossref PubMed Scopus (965) Google Scholar). Physiologically, VSMCs are contractile differentiated cells with a low ability of proliferation and apoptosis within the middle layer of vessel wall (5Li P. Zhu N. Yi B. Wang N. Chen M. You X. Zhao X. Solomides C.C. Qin Y. Sun J. MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation.Circ. Res. 2013; 113 (24014830): 1117-112710.1161/CIRCRESAHA.113.301306Crossref PubMed Scopus (136) Google Scholar). The primary functions of VSMCs are to regulate the vessel tone as well as blood pressure and flow distribution (6Yurdagul Jr., A. Finney A.C. Woolard M.D. Orr A.W. The arterial microenvironment: the where and why of atherosclerosis.Biochem. J. 2016; 473 (27208212): 1281-129510.1042/BJ20150844Crossref PubMed Scopus (91) Google Scholar). However, during the development of atherogenesis, VSMCs can switch from a contractile phenotype to a synthetic phenotype, associated with increased expression of synthetic markers, such as osteopontin (OPN), and reduced expression of contractile markers, such as α smooth muscle actin (αSMA) and smooth muscle protein 22α (SM22α) (7Gomez D. Owens G.K. Smooth muscle cell phenotypic switching in atherosclerosis.Cardiovasc. Res. 2012; 95 (22406749): 156-16410.1093/cvr/cvs115Crossref PubMed Scopus (508) Google Scholar). VSMCs can also differentiate into osteoblast-like cells that drives the process of calcification. Vascular calcification increases plaque vulnerability and exacerbates atherosclerosis progression (8Durham A.L. Speer M.Y. Scatena M. Giachelli C.M. Shanahan C.M. Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness.Cardiovasc. Res. 2018; 114 (29514202): 590-60010.1093/cvr/cvy010Crossref PubMed Scopus (334) Google Scholar). In addition, excess lipids accumulating in VSMCs can promote foam cell formation and contribute to the total intimal foam cell population in lesions (9Chaabane C. Coen M. Bochaton-Piallat M.L. Smooth muscle cell phenotypic switch: implications for foam cell formation.Curr. Opin. Lipidol. 2014; 25 (25110900): 374-37910.1097/MOL.0000000000000113Crossref PubMed Scopus (59) Google Scholar). In fact, VSMCs contribute the majority of foam cells in both human coronary atherosclerosis and advanced mouse atherosclerosis (10Allahverdian S. Chehroudi A.C. McManus B.M. Abraham T. Francis G.A. Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis.Circulation. 2014; 129 (24481950): 1551-155910.1161/CIRCULATIONAHA.113.005015Crossref PubMed Scopus (355) Google Scholar, 11Wang Y. Dubland J.A. Allahverdian S. Asonye E. Sahin B. Jaw J.E. Sin D.D. Seidman M.A. Leeper N.J. Francis G.A. Smooth muscle cells contribute the majority of foam cells in apoE (apolipoprotein E)-deficient mouse atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2019; 39 (30786740): 876-88710.1161/ATVBAHA.119.312434Crossref PubMed Scopus (111) Google Scholar). Moreover, VSMC/foam cells are potentially detrimental to plaque stability (12Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis.Acta Med. Indones. 2007; 39 (17933075): 86-93PubMed Google Scholar). TNF ligand–related molecule 1A (TL1A), also known as vascular endothelial growth inhibitor or tumor necrosis factor superfamily 15, is a type II transmembrane protein. It can be produced predominantly by endothelial cells in response to inflammatory cytokines (13Bayry J. Immunology: TL1A in the inflammatory network in autoimmune diseases.Nat. Rev. Rheumatol. 2010; 6 (20125169): 67-6810.1038/nrrheum.2009.263Crossref PubMed Scopus (32) Google Scholar, 14Zhang K. Cai H.X. Gao S. Yang G.L. Deng H.T. Xu G.C. Han J. Zhang Q.Z. Li L.Y. TNFSF15 suppresses VEGF production in endothelial cells by stimulating miR-29b expression via activation of JNK-GATA3 signals.Oncotarget. 2016; 7 (27589684): 69436-6944910.18632/oncotarget.11683Crossref PubMed Scopus (24) Google Scholar). In addition to regulation of neovascularization, high levels of TL1A were detected in human carotid atherosclerotic plaques, especially in macrophage/foam cells (15Kang Y.J. Kim W.J. Bae H.U. Kim D.I. Park Y.B. Park J.E. Kwon B.S. Lee W.H. Involvement of TL1A and DR3 in induction of pro-inflammatory cytokines and matrix metalloproteinase-9 in atherogenesis.Cytokine. 2005; 29 (15760679): 229-23510.1016/j.cyto.2004.12.001Crossref PubMed Scopus (74) Google Scholar). Furthermore, TL1A and its receptor, the death domain–containing receptor 3, participate in formation of human macrophage/foam cells by increasing uptake of oxidized low-density lipoprotein (oxLDL) and reducing cholesterol efflux in vitro (16McLaren J.E. Calder C.J. McSharry B.P. Sexton K. Salter R.C. Singh N.N. Wilkinson G.W. Wang E.C. Ramji D.P. The TNF-like protein 1A-death receptor 3 pathway promotes macrophage foam cell formation in vitro.J. Immunol. 2010; 184 (20410491): 5827-583410.4049/jimmunol.0903782Crossref PubMed Scopus (63) Google Scholar). Therefore, TL1A may be a mediator of atherosclerosis. However, whether TL1A can affect atherosclerosis in vivo remains unknown. Although apoE-deficient (apoE−/−) mice can spontaneously develop atherosclerotic lesions when they are fed chow diet, the pro-atherogenic high-fat diet (HFD)-fed apoE−/− mice have been used more frequently as the model for atherosclerosis investigation. Compared with chow diet, HFD feeding can substantially reduce the time for development of lesions. In addition, the HFD feeding can generate severe human-like hypercholesterolemia in the animals (17Ma C. Zhang W. Yang X. Liu Y. Liu L. Feng K. Zhang X. Yang S. Sun L. Yu M. Yang J. Li X. Hu W. Miao R.Q. Zhu Y. et al.Functional interplay between liver X receptor and AMP-activated protein kinase α inhibits atherosclerosis in apolipoprotein E-deficient mice-A new anti-atherogenic strategy.Br. J. Pharmacol. 2018; 175 (29394501): 1486-150310.1111/bph.14156Crossref PubMed Scopus (26) Google Scholar, 18Nakashima Y. Plump A.S. Raines E.W. Breslow J.L. Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.Arterioscler. Thromb. 1994; 14 (8274468): 133-14010.1161/01.atv.14.1.133Crossref PubMed Google Scholar). Therefore, in this study, we used HFD-fed apoE−/− mice to determine the effect of TL1A on the development of atherosclerosis and the involved mechanisms. To determine whether TL1A can influence the development of atherosclerosis, we fed apoE−/− mice the pro-atherogenic HFD for 12 weeks, a duration that can induce lesions substantially. Meanwhile, mice were intraperitoneally (i.p.) injected with PBS (control group) or recombinant human TL1A protein (TL1A group), twice a week. At the end of treatment, we collected aortic samples and determined both en face arterial and aortic root sinus lesions by Oil Red O staining. Compared with lesions in control mice (∼8.9 × 106 µm2), TL1A treatment reduced en face aortic lesions to ∼4.7 × 106 µm2 (Fig. 1A, left and middle panels). Meanwhile, in different segments of aortas (Fig. 1B), TL1A treatment inhibited the progression of lesions in descending aorta, thoracic aorta, and abdominal aorta, but not aortic arch (Fig. 1A, right panel). Furthermore, TL1A treatment substantially reduced sinus lesion size in aortic root, from 6.04 × 105 in the control group to 4.43 × 105 µm2 in the TL1A-treated group (Fig. 1C). To investigate the effect of TL1A on vascular structure, we determined collagen content and necrotic areas in aortic root cross-sections. Fig. 1D shows that TL1A substantially reduced the area of necrotic cores (∼50%) while increasing thickness of the fibrous caps (∼1.5-fold) and collagen content (∼1.5-fold) in lesion areas, indicating that TL1A enhances plaque stability. Vascular calcification can increase risk of vulnerable lesion rupture (19Finn A.V. Nakano M. Narula J. Kolodgie F.D. Virmani R. Concept of vulnerable/unstable plaque.Arterioscler. Thromb. Vasc. Biol. 2010; 30 (20554950): 1282-129210.1161/ATVBAHA.108.179739Crossref PubMed Scopus (803) Google Scholar). By completing the Alizarin Red S staining of aortic root cross-sections, we found a marked decrease of calcification-positive areas in lesion areas of TL1A-treated mice (Fig. 1E). Taken together, these results suggest that TL1A inhibits the development of atherosclerosis, enhances lesion stability, and reduces vascular calcification. To assess whether the inhibition of lesions by TL1A is related to changes of serum lipid profiles, levels of total, low-density lipoprotein (LDL)-, and high-density lipoprotein (HDL)-cholesterol and triglyceride were determined. As shown in Table 1, TL1A had little effect on lipid profiles, suggesting that the reduction of atherosclerosis is unrelated to serum cholesterol levels.Table 1TL1A treatment has little effect on serum lipid profiles and body weight gain. Male apoE−/− mice (∼8 weeks old) were randomly divided into two groups (10 mice/group) and received the treatment indicated in Fig. 1. At the end of the experiment, levels of serum total, LDL- and HDL-cholesterol and triglyceride were determinedParameterControlTL1ATotal cholesterol (mm)35.724 ± 3.66936.03 ± 4.629LDL-cholesterol (mm)12.645 ± 1.43612.925 ± 1.359HDL-cholesterol (mm)5.175 ± 0.5785.771 ± 0.959Triglyceride (mm)0.942 ± 0.0691.092 ± 0.187Body weight (g)32.73 ± 2.05132.34 ± 2.327Liver weight/body weight (%)6.103 ± 0.3886.111 ± 0.914 Open table in a new tab Although macrophage/foam cells play an important role in atherosclerosis, our results show that TL1A had little effect on monocyte/macrophage marker 2 (MOMA2) levels in aortic root (Fig. 2A). In addition, more foam cells were determined with peritoneal macrophages collected from TL1A-treated mice than controls (Fig. 2B), which is consistent with the previous report that TL1A stimulates differentiation of foam cells from either THP-1/PMA macrophages or primary human monocyte-derived macrophages in vitro (16McLaren J.E. Calder C.J. McSharry B.P. Sexton K. Salter R.C. Singh N.N. Wilkinson G.W. Wang E.C. Ramji D.P. The TNF-like protein 1A-death receptor 3 pathway promotes macrophage foam cell formation in vitro.J. Immunol. 2010; 184 (20410491): 5827-583410.4049/jimmunol.0903782Crossref PubMed Scopus (63) Google Scholar). To further confirm the effects of TL1A on formation of macrophage/foam cells, we isolated peritoneal macrophages from C57BL/6J mice and treated the oxLDL-loaded cells with TL1A or TL1A plus T0901317 (T317, a synthetic ligand for liver X receptor (LXR), can inhibit formation of macrophage/foam cells). As shown in Fig. 2C, TL1A moderately increased the number of foam cells both in the presence or absence of oxLDL, whereas the number was reduced by T317 in the presence of oxLDL. Meanwhile, TL1A antagonized T317-reduced foam cells. Both ABC transporter A1 (ABCA1) and ABCG1, two target genes of LXR, are the main molecules mediating macrophage cholesterol efflux to reduce foam cell formation and activated by T317 (17Ma C. Zhang W. Yang X. Liu Y. Liu L. Feng K. Zhang X. Yang S. Sun L. Yu M. Yang J. Li X. Hu W. Miao R.Q. Zhu Y. et al.Functional interplay between liver X receptor and AMP-activated protein kinase α inhibits atherosclerosis in apolipoprotein E-deficient mice-A new anti-atherogenic strategy.Br. J. Pharmacol. 2018; 175 (29394501): 1486-150310.1111/bph.14156Crossref PubMed Scopus (26) Google Scholar). At the molecular level, TL1A alone had moderate effects on ABCA1 expression but inhibited ABCG1 expression. In addition, TL1A substantially attenuated T317-induced ABCA1/G1 expression (Fig. 2, D and E). CD36 is a classic receptor for uptake of oxLDL by monocyte/macrophages, thereby facilitating formation of macrophage/foam cells and atherosclerosis development. CD36 can be activated by the transcription factor of peroxisome proliferator-activated receptor γ (PPARγ) (20Yang X. Zhang W. Chen Y. Li Y. Sun L. Liu Y. Liu M. Yu M. Li X. Han J. Duan Y. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) and CD36 protein expression: the dual pathophysiological roles of progesterone.J. Biol. Chem. 2016; 291 (27226602): 15108-1511810.1074/jbc.M116.726737Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), whereas sterol-responsive nuclear receptors, LXRα and LXRβ, are key determinants of cellular cholesterol homeostasis and promote cellular cholesterol efflux by up-regulating ABCA1 and ABCG1 expression (21Zhao C. Dahlman-Wright K. Liver X receptor in cholesterol metabolism.J. Endocrinol. 2010; 204 (19837721): 233-24010.1677/JOE-09-0271Crossref PubMed Scopus (311) Google Scholar). We found that although TL1A alone moderately induced expression of PPARγ and its target, CD36, in macrophages, it did not further enhance the oxLDL-induced PPARγ and CD36 levels (Fig. 2, F and G). In contrast, LXRα and LXRβ expression was decreased by TL1A treatment in the presence or absence of oxLDL (Fig. 2, F and G). Functionally, we found that TL1A reduced cholesterol efflux to HDL from macrophages (Fig. 2H). Taken together, these results suggest that TL1A promotes formation of foam cells derived from macrophages by inducing CD36 expression and inhibiting ABCA1/G1 expression. It also implies that TL1A-inhibited atherosclerosis is unrelated to the formation of macrophage/foam cells. VSMC is another main cell type making contributions to atherosclerosis by various mechanisms. For instance, the cell phenotypic switch of VSMCs plays a vital role in plaque stability (9Chaabane C. Coen M. Bochaton-Piallat M.L. Smooth muscle cell phenotypic switch: implications for foam cell formation.Curr. Opin. Lipidol. 2014; 25 (25110900): 374-37910.1097/MOL.0000000000000113Crossref PubMed Scopus (59) Google Scholar). To determine the effect of TL1A on VSMC phenotypic changes, we completed immunofluorescent staining of aortic root cross-sections with anti-αSMA and -OPN antibody. We found that expression of αSMA, the contractile marker linking to collagen content, was obviously elevated, whereas expression of OPN, the synthetic phenotype marker, was significantly reduced in TL1A-treated mice (Fig. 3A). These data indicate that TL1A can regulate the number of VSMCs in different phenotypes. We further defined the role of TL1A in VSMC phenotypic changes in vitro by treating human primary aortic smooth muscle cells (HASMCs) with TL1A. At the mRNA levels, we found that TL1A increased levels of the contractile phenotype markers, such as αSMA and SM22α, while reducing levels of OPN and epiregulin (EREG), two proliferative markers (Fig. 3B, left half). Serum response factor (SRF) and myocardin (Myocd) can form SRF-Myocd complex, and the complex functions as major transcription factors to maintain high αSMA expression and VSMCs as a contractile phenotype. The SRF-Myocd complex can be negatively regulated by Msh homeobox 1 or 2 (Msx1/2) (22Han H. Yang S. Liang Y. Zeng P. Liu L. Yang X. Duan Y. Han J. Chen Y. Teniposide regulates the phenotype switching of vascular smooth muscle cells in a miR-21-dependent manner.Biochem. Biophys. Res. Commun. 2018; 506 (30409428): 1040-104610.1016/j.bbrc.2018.10.198Crossref PubMed Scopus (8) Google Scholar). We determined that TL1A induced expression of SRF and Myocd and correspondingly reduced Msx2 expression (Fig. 3B, right half). Several studies indicate that microRNAs (miRNAs) play an important role in regulating VSMC phenotype transformation. For example, up-regulation of miR-214 by hypoxia mediates VSMC phenotypic modulation and proliferation (23Sahoo S. Meijles D.N. Al Ghouleh I. Tandon M. Cifuentes-Pagano E. Sembrat J. Rojas M. Goncharova E. Pagano P.J. MEF2C-MYOCD and leiomodin1 suppression by miRNA-214 promotes smooth muscle cell phenotype switching in pulmonary arterial hypertension.PLoS ONE. 2016; 11 (27144530): e015378010.1371/journal.pone.0153780Crossref PubMed Scopus (38) Google Scholar). miR-145, one of the most abundant VSMC miRNAs, mitigates a phenotypic switch from the contractile state into the proliferative one by inducing Myocd expression (24Cordes K.R. Sheehy N.T. White M.P. Berry E.C. Morton S.U. Muth A.N. Lee T.H. Miano J.M. Ivey K.N. Srivastava D. miR-145 and miR-143 regulate smooth muscle cell fate and plasticity.Nature. 2009; 460 (19578358): 705-71010.1038/nature08195Crossref PubMed Scopus (1243) Google Scholar). miR-29b mimics reduces cell viability and promotes SMC apoptosis by inhibiting expression of matrix metalloproteinase 2 (MMP2), one of the MMPs that enhances the VSMC switch from the contractile phenotype into the synthetic phenotype (25Shen L. Song Y. Fu Y. Li P. MiR-29b mimics promotes cell apoptosis of smooth muscle cells via targeting on MMP-2.Cytotechnology. 2018; 70: 351-35910.1007/s10616-017-0150-zCrossref PubMed Scopus (10) Google Scholar, 26Zhang J.R. Lu Q.B. Feng W.B. Wang H.P. Tang Z.H. Cheng H. Du Q. Wang Y.B. Li K.X. Sun H.J. Nesfatin-1 promotes VSMC migration and neointimal hyperplasia by upregulating matrix metalloproteinases and downregulating PPARγ.Biomed. Pharmacother. 2018; 102 (29604590): 711-71710.1016/j.biopha.2018.03.120Crossref PubMed Scopus (21) Google Scholar). In this study, we found that TL1A decreased miR-214-5p expression but increased expression of miR-145 and miR-29b (Fig. 3C, left three panels). Therefore, changes of the VSMC cell population in different phenotypes by TL1A are also related to the effect of TL1A on expression of the aforementioned miRNAs. Accumulating studies indicate that tumor suppressor p53 overexpression promotes SMC from the synthetic phenotype of migration characteristics to the quiescent and contractile phenotype by activating Myocd (27Tan Z. Li J. Zhang X. Yang X. Zhang Z. Yin K.J. Huang H. p53 promotes retinoid acid-induced smooth muscle cell differentiation by targeting myocardin.Stem Cells Dev. 2018; 27 (29482449): 534-54410.1089/scd.2017.0244Crossref PubMed Scopus (11) Google Scholar). We determined that TL1A significantly induced p53 expression in a dose-dependent manner, which was associated with decreased OPN and increased αSMA and SM22α expression proportionally (Fig. 3D). Reciprocally, when p53 expression in HASMCs was reduced by transfecting with p53 siRNA, expression of αSMA and SM22α was also markedly decreased, whereas the effect of TL1A on expression of these molecules was substantially attenuated (Fig. 3E). Taken together, the data above suggest that TL1A is able to regulate the VSMC cell population in different phenotypes and appears to enhance the switch of VSMCs from the synthetic/proliferative phenotype into the contractile phenotype by regulating expression of p53 and related miRNAs. However, the switch of VSMC phenotypes can be completed by different mechanisms. Enhancement of the apoptosis of VSMCs in the synthetic phenotype can also generate the apparent VSMC phenotype switch, particularly in the context of TL1A enhancing expression of p53 and miR-29b, two molecules that may enhance cell apoptosis. To determine the role of TL1A on cell apoptosis in vivo, we conducted TUNEL staining of the cross-sections of aortic root and thoracic aorta and quantified the number of TUNEL-positive cells. As shown in Fig. S1, TL1A had little effect on the number of apoptotic cells. Meanwhile, TL1A had little effect on expression of apoptosis-related genes, such as caspase-3, B cell lymphoma 2 (BCL2), and BCL2-associated X protein (BAX) in lesion areas either (Fig. S2). Consistent with in vivo results, we found that TL1A did not affect the viability of peritoneal macrophages (Fig. S3A) and HASMCs cultured either in normal medium or calcification medium (Fig. S3, B and C) (calcification medium can restrain cell growth; therefore, reduced cell viability was observed compared with cells cultured in normal medium). Furthermore, expression of apoptotis-related proteins was not altered by TL1A either (Fig. S3, D–G). Therefore, the results above suggest that the switch of VSMC phenotypes is not tightly linked to cell apoptosis. Similar to macrophages, VSMCs in synthetic phenotype are prone to differentiate into foam cells during atherosclerosis, and foam cells derived from VSMCs also potently contribute to the development of atherosclerosis (11Wang Y. Dubland J.A. Allahverdian S. Asonye E. Sahin B. Jaw J.E. Sin D.D. Seidman M.A. Leeper N.J. Francis G.A. Smooth muscle cells contribute the majority of foam cells in apoE (apolipoprotein E)-deficient mouse atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2019; 39 (30786740): 876-88710.1161/ATVBAHA.119.312434Crossref PubMed Scopus (111) Google Scholar). Thus, we investigated the effects of TL1A on VSMC/foam cell formation. HASMCs were treated with TL1A or TL1A plus oxLDL, followed by Oil Red O staining. Loading oxLDL to HASMCs potently increased Oil Red O–positive cells. However, TL1A clearly reduced HASMC/foam cells (Fig. 4A). Mechanistically, we determined that expression of ABCA1 and ABCG1 was increased, particularly in the presence of oxLDL. Similar to macrophages, TL1A increased CD36 and PPARγ expression in HASMCs. However, TL1A inhibited oxLDL-activated CD36 and PPARγ expression (Fig. 4, B–E). In contrast to macrophages, TL1A alone activated LXRα and LXRβ expression and further enhanced oxLDL-induced LXR expression (Fig. 4, D and E). Correspondingly, TL1A activated ABCA1 and ABCG1 expression in HASMCs and enhanced cholesterol efflux from HASMCs (Fig. 4, B, C, and F). In vivo, TL1A treatment substantially enhanced ABCG1 with little effect on ABCA1 expression (Fig. 4G). Taken together, the results above demonstrate that TL1A treatment reduces formation of foam cells derived from VSMCs mainly by enhancing ABCA1/G1 expression with LXRα/β activation and promoting cholesterol efflux. The results of Verhoeff–Van Gieson (VVG) and Alizarin Red S staining (Fig. 1, D and E) demonstrate that TL1A can enhance plaque stability, which is related to reduction of vascular calcification. To further define the functions of TL1A on vascular calcification, we conducted immunofluorescence staining to determine the expression of runt-related transcription factor 2 (RUNX2), a key transcriptional factor in osteogenic differentiation, and its downstream genes, alkaline phosphatase (ALP) and MMP9. As shown in Fig. 5 (A and B), expression of RUNX2, ALP, and MMP9 was reduced by TL1A, which was associated with reduced RUNX2 nuclear translocation. Vascular calcification is widely acknowledged as a potent risk factor for plaque rupture and the consequent sudden cardiovascular events (28Shioi A. Ikari Y. Plaque calcification during atherosclerosis progression and regression.J. Atheroscler. Thromb. 2018; 25 (29238011): 294-30310.5551/jat.RV17020Crossref PubMed Scopus (115) Google Scholar). The osteoblast-like cells deriving from VSMCs in calcified plaques plays an important role in the process of vascular calcification (29Leopold J.A. Vascular calcification: mechanisms of vascular smooth muscle cell calcification.Trends Cardiovasc. Med. 2015; 25 (25435520): 267-27410.1016/j.tcm.2014.10.021Crossref PubMed Scopus (226) Google Scholar). Therefore, we further determined the effects of TL1A on HASMC calcification in vitro. The results of Alizarin Red S staining in Fig. 5C show that calcification medium caused severe calcium accumulation within cells. However, the accumulation was clearly attenuated by TL1A. The quantification of cellular calcium content further confirms the inhibitory effect of TL1A on calcification (Fig. 5D). In addition, the results of immunofluorescent staining and Western blotting demonstrate that calcification medium induced RUNX2 expression and nuclear translocation in HASMCs, which was reduced by TL1A (Fig. 5, E–G). Consequently, calcification medium–activated expression of other critical molecules for osteoblast differentiation, such as bone morphogenetic protein 2 (BMP2), ALP, and OPN proteins in HASMCs, was reduced by TL1A (Fig. 5, H and I). Associated with changes in protein levels, mRNA levels of these osteogenic phenotype markers increased by calcification medium were attenuated by TL1A (Fig. 5J). To further confirm the direct effect of TL1A on RUNX2 activity, we constructed a RUNX2 promoter (pRUNX2). As shown in Fig. 5 (K and L), TL1A decreased calcification medium–activated RUNX2 promoter activity. It has been reported that miR-203-3p can suppress osteoblast differentiation by targeting RUNX2 (30Tu B. Liu S. Yu B. Zhu J. Ruan H. Tang T. Fan C. miR-203 inhibits the traumatic heterotopic ossification by targeting Runx2.Cell Death Dis. 2016; 7 (27787524): e243610.1038/cddis.2016.325Crossref PubMed Scopus (19) Google Scholar). Interestingly, we found that treatment of HASMCs with TL1A increased miR-203-3p expression (Fig. 3C, right), another underlying mechanism for inhibition of VSMC osteogenic differentiation by TL1A. Taken together, the results above demonstrate that TL1A inhibits vascular calcification by inactivating RUNX2 pathway both in vivo and in vitro. Chronic inflammation is a key factor in atherogenesis. It destroys the arterial wall microenvironment and contributes to plaque formation (1Wolf D. Ley K. Immunity and inflammation in atherosclerosis.Circ. Res. 2019; 124 (30653442): 315-32710.1161/CIRCRESAHA.118.313591Crossref PubMed Scopus (411) Google Scholar). Previous study has revealed that TL1A, as an important cytokine, plays a pathological role in different inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and asthma, by mediating inflammatory reactions (31Hsu H. Viney J.L. The tale of TL1A in infla