C1q tumor necrosis factor-related protein 9 in atherosclerosis: Mechanistic insights and therapeutic potential

Atherosclerosis 276 (2018) 109e116

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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Review article

C1q tumor necrosis factor-related protein 9 in atherosclerosis: Mechanistic insights and therapeutic potential Xiao-Hua Yu a, Da-Wei Zhang b, Xi-Long Zheng c, Chao-Ke Tang a, * a

Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, Medical Research Experiment Center, Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, Hunan, 421001, China b Department of Pediatrics and Group on the Molecular and Cell Biology of Lipids, University of Alberta, Alberta, Canada c Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta, T2N 4N1, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 May 2018 Received in revised form 10 July 2018 Accepted 18 July 2018 Available online 19 July 2018

C1q tumor necrosis factor-related protein 9 (CTRP9), a newly discovered adipokine, is the closest paralog of adiponectin. After proteolytic cleavage, it can release the globular domain (gCTRP9) that serves as the major circulatory isoform. Upon binding to adiponectin receptor 1 (AdipoR1) and N-cadherin, CTRP9 can activate a variety of signaling pathways to regulate glucose and lipid metabolism, vascular relaxation and cell differentiation. Circulating CTRP9 levels are significantly decreased in patients with coronary atherosclerosis disease. Data obtained from in vitro experiments and animal models suggest that CTRP9 exerts an atheroprotective effect by altering multiple pathological processes involved in atherosclerosis, including inflammation, foam cell formation, endothelial dysfunction, insulin resistance, and vascular smooth muscle cell dedifferentiation, proliferation and migration. In this review, we summarize the latest advances regarding the roles of CTRP9 in atherosclerosis with an emphasis on its potential as a novel therapeutic target in cardiovascular disease. © 2018 Elsevier B.V. All rights reserved.

Keywords: CTRP9 AdipoR1 N-cadherin Atherosclerosis Adiponectin

1. Introduction Cardiovascular disease (CVD) is the major cause of morbidity and mortality in the developed countries. Epidemiological studies have suggested that CVD accounts for >17 million deaths globally every year, which is predicted to rise to >23 million by 2030 [1].

Abbreviations: CVD, cardiovascular disease; CTRPs, C1q tumor necrosis factorrelated proteins; AMI, acute myocardial infarction; HMW, high molecular weight; AMPK, adenosine monophosphate-activated protein kinase; eNOS, endothelial nitric oxide synthase; AdipoR, adiponectin receptor; ECs, endothelial cells; MSCs, mesenchymal stem cells; SIRT1, sirtuin 1; PGC-1a, peroxisome proliferatoractivated receptor-coactivator-1a; NF-kB, nuclear factor-kB; ACC, acetyl-CoA carboxylase; LXRa, liver X receptor a; ABCA1, ATP-binding cassette transporter A1; VSMCs, vascular smooth muscle cells; ERK, extracellular signal-regulated kinase; MMP-9, matrix metalloproteinase-9; Nrf2, nuclear factor erythroid-derived 2-like 2; CAD, coronary atherosclerosis disease; HDL-C, high-density lipoprotein cholesterol; TNF-a, tumor necrosis factor-a; IL-6, interleukin-6; T2DM, type 2 diabetes mellitus; ICAM-1, ntracellular adhesion molecule-1; VCAM-1, vascular cellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1; ox-LDL, oxidized low-density lipoprotein; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; PDGF-BB, platelet-derived growth factor-BB. * Corresponding author. E-mail address: [email protected] (C.-K. Tang). https://doi.org/10.1016/j.atherosclerosis.2018.07.022 0021-9150/© 2018 Elsevier B.V. All rights reserved.

Atherosclerosis has long been thought to be the common pathological basis of most cardiovascular diseases such as myocardial infarction, stroke, and peripheral arterial disease. The formation of atherosclerotic lesions is a chronic process involving a complicated signaling network and various effector molecules [2,3]. Although statin monotherapy or its combination with other drugs has obtained considerable improvement in clinical outcomes of CVD patients, the residual risk is still a major challenge [4]. Thus, a more detailed understanding of the roles of key molecules in atherogenesis is essential to develop novel therapeutic strategies for CVD. Adipokines are bioactive substances secreted by adipose tissue, including adiponectin, leptin, omentin, and C1q tumor necrosis factor-related proteins (CTRPs). Among them, adiponectin is the most extensively investigated adipokine and confers a variety of beneficial effects on cardiovascular system [5]. As the paralogs of adiponectin, CTRPs are a highly conserved family containing 15 members from CTRP1 to CTRP15 [6,7]. Of all members, CTRP9, a secreted glycoprotein discovered in 2009, has the highest similarity to adiponectin [8]. Several lines of evidence have shown that CTRP9 can attenuate myocardial ischemia-reperfusion injury, inhibit adverse cardiac remodeling after acute myocardial infarction (AMI), and ameliorate pulmonary arterial hypertension in rodents [9e11].

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Recently, accumulating studies reveal that CTRP9 is protective against atherosclerosis through multiple mechanisms including inhibition of inflammatory response, regulation of lipid metabolism, and amelioration of endothelial dysfunction, suggesting this adipokine as a novel and promising target to prevent and treat atherosclerosis-associated disease [12,13]. In this review, we summarized the current knowledge about the roles of CTRP9 in atherosclerosis to provide a rationale for future investigation and therapeutic intervention. 2. Structural features and post-translational modifications of CTRP9 Both human and mouse CTRP9 genes have similar exon and intron structures [8]. Human CTRP9 gene (12.6 kb) contains 4 exons and is located on chromosome 13q12.12. Mouse CTRP9 gene (12.7 kb) also consists of 4 exons and is mapped to chromosome 14. Human CTRP9 protein contains 333 amino acid residues with a predicted molecular mass of 32 kDa. Similar to adiponectin, CTRP9 is composed of a signal peptide (residues 1e19) to direct protein secretion, a short N-terminal domain (residues 20e28), a collagenlike domain with 56 Gly-X-Y repeats (residues 29e196), and a Cterminal globular C1q domain (residues 197e333, Fig. 1). These 4 domains also exist in other CTRPs except CTRP4. However, CTRP9 shares the highest degree of amino acid identity (54%) with adiponectin in the C-terminal globular C1q domain, suggesting that both adipokines may have similar functions [8]. CTRP9 is also evolutionarily highly conserved among dogs, chickens, zebrafishes, frogs, mice, and humans. There are 22 Gly-X-Y repeats and one consensus GXKG(E/D) motif in the collagen-like domain of adiponectin. It has been reported that proline residues in its Gly-X-Y repeats undergo hydroxylation for structural stabilization [14,15]. Lysine residues in the consensus GXKG(E/D) motif are hydroxylated and subsequently glycosylated with a glucosyl-galactosyl group, leading to enhancement of its function [16,17]. CTRP9 has a longer collagenlike domain with 56 Gly-X-Y repeats. In line with adiponectin, mass spectrometry showed that 10 of the 13 proline residues in the

Gly-X-Y repeats of CTRP9 are hydroxylated, and two of the seven lysine residues in its consensus GXKG(E/D) motif are hydroxylated and glycosylated [8]. However, the effects of these posttranslational modifications on CTRP9 structure and function remain largely unknown. Future research in this area will help deepen the understanding of its role in cardiovascular system. Proteolysis, a common post-translational modification, frequently occurs in the CTRPs. For example, furin mediates endogenous cleavage of full-length CTRP12 to liberate the globular domain with different structure and function [18]. Similarly, CTRP9 monomers form the trimeric complexes, which undergo proteolytic cleavage to release their globular domain (gCTRP9) as the major circulatory isoform (Fig. 2) [19]. Moreover, gCTRP9 has stronger effects on the activation of adenosine monophosphate-activated protein kinase (AMPK), Akt and endothelial nitric oxide synthase (eNOS) than fulllength CTRP9 [19]. Thus, promoting gCTRP9 production may be an effective approach for enhancing the biological function of CTRP9.

3. CTRP9 expression profile CTRP9 is predominantly produced by adipocytes, but other types of cells, such as cardiomyocytes, can also synthesize this molecule [20]. This raises an intriguing possibility that CTRP9 is not only an adipokine but also a cardiokine. In accordance with adiponectin, higher levels of CTRP9 transcripts are found in female

Fig. 2. A schematic showing CTRP9 isoform production. CTRP9 is first synthesized as a monomer. Three monomers are assembled into a trimeric complex. These trimers are proteolytically cleaved to form gCTRP9, the primary circulatory isoform.

Fig. 1. Schematic of human adiponectin and CTRP9 monomer structure. Human adiponectin and CTRP9 monomers are composed of four regions: a signal peptide, an N-terminal domain, a collagen-like domain with multiple Gly-X-Y repeats, and a Cterminal globular domain homologous to the immune complement C1q. These two adipokines share 54% amino acid identity in their globular domains.

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mice than in male mice, suggesting a gender-biased expression pattern [8]. This differential expression may be associated with different sex hormones. Future studies will be needed to clarify how estrogen and androgen modulate CTRP9 expression. In leptindeficient obese ob/ob mice, CTRP9 expression is elevated in adipose tissue and the circulation at 8 weeks of age compared with lean controls, but returns to the normal levels at 12 weeks of age [8]. Increased CTRP9 levels in these young mice may represent a compensatory response prior to the development of metabolic syndrome. Genetic variation has been frequently reported in the adiponectin gene [21,22], but it is unclear whether the polymorphisms of CTRP9 gene are also present with effects on its expression. 4. CTRP9 receptors and signaling Adiponectin receptor 1 (AdipoR1), a transmembrane protein with seven helices, serves as the receptor of CTRP9 besides adiponectin. Accumulating evidence indicates that CTRP9 signaling is transduced via the AdipoR1 in RAW 264.7 macrophages [23], endothelial cells (ECs) [24], and cardiac myocytes [25]. N-cadherin, an integral membrane protein that contains five extracellular cadherin domain repeats, one transmembrane domain and one highly conserved cytoplasmic domain, is a cell surface marker of mesenchymal stem cells (MSCs) [26]. Recently, Yan et al. observed that CTRP9 is colocalized and directly interacts with N-cadherin in adipose-derived MSCs [27]. Importantly, knockout of N-cadherin but not AdipoR1 completely abrogates the effects of CTRP9 on adipose-derived MSCs, suggesting that N-cadherin is a novel and specific CTRP9 receptor [27]. CTRP9 signal transduction appears to be complex with the

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involvement of numerous downstream molecules (Fig. 3). AMPK, a serine/threonine kinase sensitive to cellular energy alternation, acts as a major downstream component of CTRP9 signaling. It has been reported that CTRP9 triggers the AdipoR1/SIRT1 (sirtuin 1)/PGC-1a (peroxisome proliferator-activated receptor-coactivator-1a) signaling and then phosphorylates AMPK in ECs [24,28]. It is well known that nuclear factor-kB (NF-kB) is a transcriptional factor stimulating the production of pro-inflammatory cytokines [29]. Treatment with recombinant CTRP9 protein activates the AMPK/ ACC (acetyl-CoA carboxylase) signaling, leading to inhibition of NFkB nuclear translocation and pro-inflammatory cytokine secretion in human aortic ECs [30]. Liver X receptor a (LXRa) is known to play a central role in stimulating the gene transcription of ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG1 [31,32]. Activation of AMPK by CTRP9 markedly increases their expression in an LXRa-dependent manner in vascular smooth muscle cells (VSMCs) [33]. Notably, the vasodilation effect of CTRP9 is also mediated by the AMPK/Akt/eNOS pathway [34]. Upon binding to N-cadherin, CTRP9 triggers extracellular signal-regulated kinase (ERK)1/2 phosphorylation, leading to a significant increase in the expression of matrix metalloproteinase-9 (MMP-9) and nuclear factor erythroid-derived 2-like 2 (Nrf2) in adipose-derived MSCs [27]. Additionally, CTRP9 increases the expression and activity of zinc finger transcription factor GATA4 in an ERK5-dependent manner in mouse cardiomyocytes [35]. 5. The anti-atherogenic action of CTRP9 Adiponectin, CTRP1, and CTRP3 have been shown to participate in atherosclerosis development. As the closest paralog of adiponectin among CTRPs, CTRP9 is also tightly associated with

Fig. 3. CTRP9 signaling transduction. CTRP9 has two receptors: AdipoR1 and N-cadherin. Upon binding to AdipoR1, CTRP9 activates AMPK through the SIRT1/PGC-1a signaling, leading to inhibition of NF-kB in an ACCdependent manner and upregulation of LXRa expression. AMPK can also promote eNOS production by activating Akt. The effects of CTRP9 on GATA4 are mediated possibly by the AdipoR1/ERK5 pathway. In addition, CTRP9 interacts with N-cadherin to phosphorylate ERK1/2, resulting in increased expression of MMP-9 and Nrf2.

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atherosclerosis. For instance, male adult mice display a significant decrease in serum CTRP9 levels and adipocyte CTRP9 expression after AMI [10]. A similar result is also observed in mice with myocardial ischemia-reperfusion injury [9], and in subjects with coronary atherosclerosis disease (CAD) [36]. Moreover, serum CTRP9 levels are positively correlated with high-density lipoprotein cholesterol (HDL-C) levels in these CAD patients, suggesting that CTRP9 may be a potential biomarker of atherosclerosis [36]. Consistently, overexpression of CTRP9 protects mice from dietinduced obesity, a well-known risk factor for atherosclerosis [37]. Injection of an adenoviral vector expressing CTRP9 via mouse jugular vein dramatically inhibits neointimal formation after left femoral arterial injury [38]. It is well known that the stable plaques can transform into vulnerable plaques prone to rupture. Once ruptured, acute cardiovascular events occur due to thrombus formation [39]. Enhanced platelet activity is a leading cause of atherothrombosis [40]. In a recent study, pretreatment with CTRP9 markedly prevents adenosine diphosphate (ADP)-stimulated platelet activation in mouse platelet samples [41]. In apoEe/e mice fed a high-fat diet and receiving constrictive collars on the right carotid artery, lentivirus-mediated exogenous expression of CTRP9 was shown to decrease macrophage numbers and lipid content but increase VSMC numbers and collagen content within the mature carotid plaques, suggesting CTRP9 as a positive regulator of the plaque stability [42]. 6. Potential mechanisms underlying the atheroprotection of CTRP9 Atherogenesis, a complex and multifactorial process, involves inflammation, dysregulation of lipid metabolism, endothelial dysfunction, insulin resistance, and VSMC phenotypic switch and subsequent proliferation and migration [43e45]. As a unique and pleiotropic adipokine, CTRP9 protects against the development of atherosclerosis through multiple mechanisms (Fig. 4). 6.1. CTRP9 inhibits vascular inflammation Atherosclerosis is a maladaptive, nonresolving chronic inflammatory disorder occurring at the sites with blood flow disturbance.

T cells are known to trigger the inflammatory response at these sites by secreting a variety of pro-inflammatory mediators [46]. Macrophages, important effector cells in atherogenesis, are subdivided into the classical M1 and alternative M2 categories. M1 macrophages produce pro-inflammatory cytokines, while M2 macrophages possess anti-inflammatory properties [47,48]. The atheroprotective action of regulatory T cells (Tregs) is also associated with their ability to secrete anti-inflammatory cytokines [49]. Thus, the imbalance between pro- and anti-inflammatory signals in favor of the former is a major driving force of atherosclerosis at all stages from lesion formation to plaque destabilization and eventual rupture. Like its paralog adiponectin, CTRP9 participates in the regulation of inflammatory response. Circulating CTRP9 levels are significantly decreased in CAD patients with an increase in serum tumor necrosis factor-a (TNF-a) and interleukin (IL)-6 concentrations, demonstrating an inverse correlation [36]. Therefore, CTRP9 may serve as a novel molecular marker to reflect inflammatory status associated with atherosclerosis. However, another study showed that patients with type 2 diabetes mellitus (T2DM) and CAD display a significant increase in serum CTRP9, TNF-a and IL-6 levels, suggesting a positive correlation between CTRP9 and TNFa and IL-6 [50]. Given that deletion of CTRP9 in mice attenuates insulin sensitivity [51], elevated levels of CTRP9 may represent a compensatory response to insulin resistance and hyperglycemia in these patients. Further studies will be needed to confirm this possibility. The inflammatory response in blood vessels results from a complex interaction between circulating leukocytes and vascular endothelium. During atherogenesis, circulating monocytes transmigrate into the subintima where they differentiate into macrophages [52]. This process is mediated predominantly by intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) [53]. It has been reported that CTRP9 significantly decreases the levels of these three pro-inflammatory mediators and consequently inhibits TNF-a-induced adhesion of monocytes to human aortic ECs [30]. CTRP9-mediated prevention of THP-1 monocyte adhesion to VSMCs is also associated with downregulation of ICAM-1 and VCAM-1 expression [33]. In RAW

Fig. 4. The role of CTRP9 in atherosclerosis. CTRP9 exerts a significant protective effect on atherosclerosis, which involves multiple factors and pathways. On one hand, CTRP9 maintains beneficial lipid profile, promotes plaque stability, and increases insulin sensitivity. On the other hand, it ameliorates endothelial dysfunction, inhibits transformation of VSMCs into macrophage-like cells, mitigates inflammatory response, hinders VSMC proliferation and migration, reduces foam cell formation, and suppresses thrombosis.

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264.7 macrophages treated with oxidized low-density lipoprotein (ox-LDL), administration of recombinant gCTRP9 protein markedly attenuates TNF-a and MCP-1 production via the AdipoR1/AMPK/ NF-kB signaling pathway [23]. Moreover, overexpression of CTRP9 in apoEe/e mice increases carotid plaque stability by inhibiting the secretion of TNF-a and MCP-1 from macrophage [42]. Thus, stimulation of CTRP9 biogenesis and supplementation with exogenous CTRP9 could be valuable approaches to mitigate vascular inflammation and stabilize atherosclerotic plaques. 6.2. CTRP9 regulates lipid metabolism Dyslipidemia plays a central role in the occurrence and development of atherosclerosis. HDL-C exerts an atheroprotective effect, but low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) are proatherogenic. Epidemiological studies have clearly shown that a 1 mg/dL increase in HDL-C is associated with a significant reduction of coronary heart disease risk in both men (2%) and women (3%) [54]. In CAD patients, circulating CTRP9 concentrations are positively correlated with serum HDL-C levels [36]. Importantly, injection of recombinant gCTRP9 increases serum HDL-C levels but decreases LDL-C and TG levels in teleost [55]. CTRP9 transgenic mice lacking apoE exhibit a significant reduction in carotid plaque lipid contents, suggesting a protective role for CTRP9 in controlling lipid accumulation within the intima [42]. In mice fed a high-fat diet, CTRP9 overexpression stimulates AMPK phosphorylation and autophagy, leading to inhibition of endoplasmic reticulum stress and the subsequent alleviation of hepatic steatosis [56]. All of these data suggest that CTRP9 contributes to the maintenance of favorable lipid metabolism. Foam cell formation in the arterial intima is a major hallmark of early-stage lesions of atherosclerosis, resulting from the uncontrolled uptake of modified lipoprotein particles and impaired intracellular cholesterol export [57]. Scavenger receptor class A (SRA), CD36, and lectin-like oxidized low-density lipoprotein receptor1 (LOX-1) play a central role in the uptake of modified lipoprotein particles by the arterial wall cells [58]. ABCA1 and ABCG1 are primarily responsible for intracellular cholesterol export [59,60]. VSMCs are an important origin of foam cells, comprising at least 50% of total foam cells in human coronary atherosclerotic plaques [61]. A recent study showed that CTRP9 decreases LOX-1 expression in cholesterol-loaded VSMCs with a significant increase in the levels of ABCA1 and ABCG1 [33]. Thus, CTRP9 may play an important role in inhibiting the transformation of VSMCs into foam cells. 6.3. CTRP9 ameliorates endothelial dysfunction The endothelium is a monolayer of ECs in the inner surface of the vascular lumen and plays an important role in maintaining vascular tone, blood fluidity and permeability as well as protecting against thrombosis [62,63]. Endothelial dysfunction usually occurs after exposure to detrimental stimuli including shear stress and oxidative stress [64,65]. Like vascular inflammation and lipid accumulation, endothelial dysfunction is also an essential driver of atherosclerosis development [66,67]. Nitric oxide (NO), a potent vasodilator, is produced from Larginine by the eNOS. Impairment of endothelium-dependent vasodilatation due to decreased NO production is an important feature of endothelial dysfunction and precedes atherosclerotic lesion formation [68]. In aortic rings isolated from wild-type C57BL/ 6 mice, CTRP9 facilitates NO release and consequently stimulates vascular relaxation through the AdipoR1/AMPK/Akt/eNOS pathway with an effect exceeding that of adiponectin by approximately three-fold [69]. Pretreatment with recombinant CTRP9 protein also

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increases eNOS-dependent NO synthesis in human umbilical vein endothelial cells (HUVECs) in the presence of ox-LDL [28]. In addition to NO, ECs can secrete several other vasodilators, such as bradykinin, and the vasoconstrictors, such as angiotensin II (Ang II) and endothelin-1 (ET-1), to maintain the appropriate vascular contractility [70]. Notably, overexpression of CTRP9 also activates the PI3K (phosphatidylinositol 3-kinase)/Akt pathway to prevent ET-1 secretion in human pulmonary arterial ECs [11], suggesting that CTRP9 serves as a novel vasorelaxative adipokine by influencing NO and ET-1 production. However, it is not yet known whether CTRP9 has a regulatory effect on bradykinin and Ang II expression. Reduced ECs resulting from excessive apoptosis has long been considered as an important cause to disrupt the structural and functional integrity of the endothelium [71]. It has been reported that CTRP9 decreases apoptotic incidence in HepG2 cells [56], keratinocytes [72], adipose-derived MSCs [27], and cardiomyocytes [73]. Consistently, pretreatment with recombinant CTRP9 protein inhibits ox-LDL-induced apoptosis of HUVECs [28]. Overexpression of CTRP9 also reduces apoptosis in human pulmonary arterial ECs by activating the PI3K/Akt signaling [11]. Thus, blocking EC apoptosis is another important mechanism by which CTRP9 protects against endothelial dysfunction. Oxidative stress, a disturbed redox homeostasis with excessive reactive oxygen species (ROS) production and impaired antioxidant defense system, causes oxidative damage of cells and tissues. Accumulating evidence indicates that oxidative stress is one of the triggers of endothelial dysfunction and atherosclerosis [74,75]. Of note, CTRP9 is also involved in regulation of oxidative stress. Kambara et al. showed that mice lacking CTRP9 display a significant increase in cardiac ROS content [25]. In contrast, CTRP9 overexpression alleviates oxidative damage in diabetic mouse heart [20]. Incubation of HUVECs with CTRP9 reduces ox-LDL-induced ROS production [28]. Administration of exogenous CTRP9 increases the expression and activities of antioxidant enzymes manganese-dependent superoxide dismutase (MnSOD) and isocitrate dehydrogenase 2 (IDH2) through the AdipoR1/SIRT1/PGC-1a pathway in human aortic ECs exposed to high glucose [24]. Collectively, these observations suggest that CTRP9 functions as an antioxidative adipokine and targeting CTRP9 may have enormous potential for ameliorating oxidative stress-associated endothelial dysfunction. 6.4. CTRP9 blocks transdifferentiation of VSMCs into macrophagelike cells Macrophages have long been considered as a critical component in atherosclerotic lesions. The majority of macrophages in the arterial intima are derived from circulating monocytes. However, several lines of evidence have demonstrated that approximately 40% of macrophages in advanced human atherosclerotic plaques are in fact from VSMCs [61,76]. The transition of VSMCs to macrophage-like cells not only impairs vascular contractile function but also promotes cellular proliferation and migration and proinflammatory mediator secretion [77]. Blockade of this conversion is known to inhibit neointimal lesion formation in animal models of carotid artery ligation [78,79]; however, promoting this process accelerates atherosclerosis development in mice [80]. Abnormal VSMC phenotypic switch is thus thought to be an important contributor to atherogenesis. The addition of CTRP9 to cholesterolloaded VSMCs was shown to decrease macrophage marker CD68 levels but increase the levels of VSMC dedifferentiation markers, such as smooth muscle a-actin, suggesting CTRP9 as a novel modulator of VSMC transdifferentiation into macrophage-like cells at least in in vitro models [33].

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6.5. CTRP9 decreases VSMC proliferation and migration Under physiological conditions, VSMCs locate in the media of arterial wall and maintain a non-proliferative quiescent status. In response to pathological stimuli, VSMCs acquire considerably proliferative capacity and migrate from the media to the intima, thereby promoting neointimal hyperplasia and atherosclerotic lesion formation [81]. In addition to VSMC phenotypic switch, CTRP9 participates in the regulation of VSMC proliferation and migration. In hypoxia-treated human pulmonary arterial smooth muscle cells (HPASMCs), administration of recombinant CTRP9 protein leads to a significant reduction in cellular proliferative and migratory potentials by inhibiting the TGF-b1 (transforming growth factor-b1)/ERK1/2 signaling axis [82]. CTRP9 also interferes with DNA synthesis and decreases the number of migratory cells in response to platelet-derived growth factor-BB (PDGF-BB) in human VSMCs [38]. Mechanistically, CTRP9 activates the cAMP (cyclic adenosine monophosphate)/PKA (protein kinase A) pathway, leading to inhibition of PDGF-BB-induced ERK1/2 phosphorylation. Moreover, delivery of an adenoviral vector expressing CTRP9 through mouse jugular vein suppresses cell proliferation and attenuates neointimal formation in response to arterial wire injury [38], suggesting a therapeutic potential for CTRP9 in vascular restenosis after angioplasty. 6.6. CTRP9 enhances insulin sensitivity Insulin resistance is a critical etiology for T2DM that is characterized by excessive glucose production in the liver and hyperglycemia. T2DM is a well-documented risk factor for subclinical atherosclerosis [83,84]. Like its role in lipid metabolism, CTRP9 is associated with glucose metabolism. In patients with prediabetes or T2DM, circulating CTRP9 levels are significantly decreased with a negative correlation with fasting blood glucose concentrations [85]. On the other hand, serum CTRP9 levels are higher in subjects with impaired glucose tolerance or newly diagnosed T2DM when compared to healthy controls [86]. The individuals with obesity, a major public health problem worldwide, are at higher risk of metabolic syndrome, T2DM, and CVD [87]. It was reported that circulating CTRP9 levels are elevated in obese patients but decreased following weight loss surgery [88]. Higher CTRP9 levels in the context of T2DM or obesity may be a compensatory response that is similar to insulin. Of importance, loss of CTRP9 causes insulin resistance in the peripheral tissues and attenuates insulin tolerance in mice [51]; however, CTRP9 transgenic mice display an obvious improvement in insulin resistance and metabolic profile with reduced plasma levels of fasting insulin and glucose [37]. These animal models reveal a beneficial role for CTRP9 in controlling insulin resistance, although the underlying mechanisms remain to be determined. Targeting this adipokine may represent a novel strategy to increase insulin sensitivity and treat T2DM-associated atherosclerosis. 7. The therapeutic strategies to promote CTRP9 production CTRP9 exerts its biological effects in an autocrine, paracrine or endocrine manner. Given its atheroprotective properties, enhancement of circulating CTRP9 levels could be a novel and promising therapeutic approach for the atherosclerosis-associated disease. It is still unfeasible for the conversion of recombinant CTRP9 protein to a viable drug at present. Thus, the strategy aiming to stimulate endogenous CTRP9 production may be more valuable. Dysregulation of microRNAs (miRNAs) is closely associated with atherosclerosis development [89]. Administration of aroclor 1260, a persistent organic pollutant contributing to multiple vascular

diseases such as atherosclerosis, has been shown to increase miR31 levels in HUVECs [90]. Importantly, elevated miR-31 expression is observed in the arterial wall isolated from arteriosclerosis obliterans subjects, and transfection with miR-31 mimics promotes VSMC proliferation and migration in response to PDGF-BB via silencing of mitofusin-2 [91]. Overexpression of miR-31 also induces macrophage apoptosis and accelerates arteriosclerosis in apoEe/e mice by directly targeting NADPH oxidase 4 [92]. Interestingly, CTRP9 is also identified as one target gene of miR-31 [93]. Thus, in vivo delivery of miR-31 inhibitors contributes to CTRP9 upregulation and may have a potential therapeutic benefit for CVD patients. CTRP9 is known to act through AdipoR1 and N-cadherin. Recently, Okada-Iwabu et al. synthesized an orally active smallmolecule AdipoR1/2 agonist, termed AdipoRon [94]. They found that administration of AdipoRon ameliorates insulin resistance and prolongs the shortened lifespan in a mouse model of T2DM [94]. Two short peptides Pep70 and ADP 355 have also been reported to directly activate AdipoR1 [95,96]. N-cadherin functions via a homophilic and calcium-dependent mechanism and the homophilic binding site resides in its first extracellular cadherin domain (ECD1) [97]. Two cyclic peptides INPISGQ and HAVDI are highly specific and potent antagonists of N-cadherin by binding to the ECD1 [98,99]. However, a dimeric version of these two peptides can activate N-cadherin and consequently promote neurite outgrowth in a similar manner to native N-cadherin [100]. Therefore, stimulating CTRP9 function with its receptor agonists could be used therapeutically to inhibit atherosclerosis. 8. Conclusion and future directions CTRP9 has attracted a lot of attention since it was discovered in 2009. This adipokine is involved in multiple pathophysiological processes associated with atherosclerosis. Although the vast majority of studies have shown beneficial effects, a recent report revealed a paradoxical result that serum CTRP9 levels positively correlate with arterial stiffness in T2DM patients [101]. Thus, the exact role of CTRP9 in atherosclerosis still needs to be determined. Whether circulating CTRP9 can be used as a potential biomarker also requires more careful evaluation. Treatment with recombinant CTRP9 protein was shown to reduce TG biogenesis induced by sterol regulatory element-binding protein 1 in HepG2 cells [56], while it remains largely unknown whether this adipokine also contributes to its clearance. Given the critical role of hepatic lipase and lipoprotein lipase in the process of TG degradation [102], future research will also need to focus on the effects of CTRP9 on expression and activity of these lipases. Very little is known about the mechanisms underlying CTRP9 modulation. Identification of key regulators is essential to develop CTRP9-based therapies and may provide novel insights into its physiological functions. In addition, the following issues need to be addressed. Does N-cadherin serve as a receptor of CTRP9 in macrophages, VSMCs and ECs, the major effector cells involved in atherosclerosis? If so, its agonists can be developed as a therapeutic agent for the atherosclerosis-associated disease. Are there any other specific receptors for CTRP9 besides N-cadherin? How is CTRP9 synthesized and secreted by adipocytes? What is the intracellular trafficking itinerary of CTRP9? Answers to these questions will provide insightful knowledge about the roles of CTRP9 in atherosclerosis and help develop novel molecule-targeted drugs. Conflicts of interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

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