AMI: Acute Myocardial Infarction; ANGPTL4: Angiopoietin-like Protein 4; C5a: Component 5a; cANGPTL4: c-terminal ANGPTL4; LPL: Lipoprotein Lipase; nANGPTL4: n-terminal ANGPTL4

Angiopoietin-like 4 (ANGPTL4) is a multifaceted secreted protein discovered by three different institutions in 2000 simultaneously [1]. It is expressed in adipose tissues, liver, muscle, heart, kidney, skin, and other tissues. The nutritional, metabolic, and inflammatory status of the organism regulate the expression of ANGPTL4 [2-4]. ANGPTL4 was initially found to inhibit lipoprotein lipase (LPL) activity thereby regulating the triglyceride levels [5-8]. Human genetic studies have shown that genetic inactivation of ANGPTL4 could reduce the risk of diabetes and coronary artery disease progression significantly [7-10]. Unfortunately, lacking ANGPTL4 in mice consumed dietary saturated fat induces a pro-inflammatory and ultimately lethal phenotype, including fibrinopurulent peritonitis, and ascites [11]. Antibodies against ANGPTL4 result in lymphadenopathy [9] and ascites in mice or monkeys [12]. These unexpected reports led us to focus on the role of ANGPTL4 in inflammatory processes [13]. While extensive research portray ANGPTL4 as an inflammatory mediator [14-18], there is a considerable body of evidence ascribing that ANGPTL4 protects against the severe pro-inflammatory effects of saturated fat and increases the number of anti-inflammatory macrophages in peritonitis and myocardial infarction (AMI) [11,19]. ANGPTL4’s role in inflammation appears to be bidirectional, and its exact mechanism is not fully understood.

In a recent issue, we systematically reviewed the functions, potential underlying mechanisms, and therapeutic value of ANGPTL4 in inflammation, including lung injury, cardiovascular disease, pancreatitis, gastrointestinal disease and metabolic disorders [20]. ANGPTL4 has been reported to be involved in inflammation processes of acute pulmonary diseases including influenza pneumonia and LPS-induced acute lung injury, as well as chronic pulmonary diseases such as chronic obstructive pulmonary disease. ANGPTL4 expression is directly upregulated by influenza infection through the IL6-STAT3 signaling cascade. Meanwhile, cleavage of full-length ANGPTL4 (fANGPTL4) to c-terminal ANGPTL4 (cANGPTL4) due to furin activation results in severe lung injury characterized by extensive pulmonary hemorrhage and immune cell infiltration [16]. In both lung tissue from an acute injury mouse model and LPS-treated human alveolar epithelial cells, ANGPTL4 expression significantly increases and is positively correlated with the inflammation in lung tissue (TNF-α, IL-6, and neutrophil infiltration) [14,21]. Patients suffering from chronic obstructive pulmonary disease often have upregulated levels of circulating ANGPTL4, which are associated with pulmonary function and systemic inflammation [22].

In addition to lung injury, ANGPTL4 also plays a crucial role in the development and progression of pancreatitis. Variations in the lipoprotein lipase pathway regulatory genes, including LPL, APOC3, APOA5, ANGPTL3, and ANGPTL4, that increase plasma triglyceride levels, are associated with a higher risk of acute pancreatitis [23]. In the microarray analysis, ANGPTL4 exhibits the most pronounced up-regulation among genes in pancreatic tissues from both mild and severe acute pancreatitis mice models. Macrophage activation and infiltration into the pancreas are enhanced by elevated ANGPTL4 levels, leading to increased complement component 5a (C5a) through the PI3K/AKT signaling pathway. C5a receptor activation results in hypercytokinemia, which advances pancreatitis by accelerating acinar cell damage [17]. In conclusion, targeting ANGPTL4 is considered a prospective therapeutic approach for pancreatitis.

Cho et al. [19] proved that in a co-culture environment with macrophages, mesenchymal stem cells actively expressed ANGPTL4, which in turn attenuated the macrophage polarization towards the pro-inflammatory phenotype. In animal models of peritonitis and myocardial infarction, injection of ANGPTL4 protein significantly enhanced the anti-inflammatory effects of macrophage infiltration. Post-acute myocardial infarction myocardial reperfusion injury encompasses a sequence of pathological responses, including hemorrhage, hematoma formation, and inflammatory responses. Hypoxia-induced ANGPTL4 expression may modulate vascular injury, infarct size, and the occurrence of no-reflow in AMI [24]. Lee et al. [25] invented a paintable and adhesive hydrogel patch that could encapsulate and effectively release the anti-inflammatory protein ANGPTL4 into the infarcted heart to alleviate the inflammatory response. Heart tissues receiving ANGPTL4-loaded hydrogel patches exhibited the reduced presence of inflammatory macrophages and cytokines (IL-1β, IL-6, and TNF-α), enhanced vascularization and structural cardiomyocyte maturation were observed in heart tissues received ANGPTL4-loaded hydrogel patches [26]. Furthermore, ANGPTL4 serum levels could be used to predict the occurrence of no-flow in ST-elevation myocardial infarction patients after successful percutaneous coronary intervention [24].

Numerous gastrointestinal diseases manifest a prolonged and intensified inflammation, which can result in substantial tissue damage following hypercykinaemia. Dextran sulfate sodium treated ANGPTL4^-/- mice exhibit an exacerbated chronic inflammation, as well as the enrichment of genes associated with leukocyte migration and infiltration, which resembles the patterns observed in inflamed ulcerative colitis. In human colon epithelial cells, ANGPTL4 upregulated tristetraprolin expression to mediate the chronic inflammatory response [18]. In the intestinal mucosa of inflammatory bowel disease patients, endoscopic inflammation was found to modulate bile acid-inducible microbial genes in the microbiota, which in turn regulate the intestinal inflammatory responses via ANGPTL4 [27].

Atherosclerosis, diabetes, and obesity are regarded as chronic low-grade inflammatory diseases, which are persistent, non-specific inflammatory conditions characterized by increased concentrations of C-reactive protein, TNF-α, IL-1 [28], and activations of immune cells [29]. Tong et al. [30] demonstrated that the ANGPTL4 gene SNP1044250 independently contributes to the development of metabolism syndrome when body weight increases. The impact of ANGPTL4 on atherogenesis appears to employ both positive and negative effects depending on the context [9,31-34]. Cho et al. [35] discovered the positive effect of ANGPTL4 on vascular stability and inflammation in atherosclerosis. ANGPTL4 treatment suppressed the phenotypic transformation of smooth muscle cells into macrophage-like and foam-like cells through direct KLF4 downregulation or by diminishing the NOX1 activation of KLF4. The thickness of fibrous caps and the number of SM22α(+), SMA(+), and SM-MHC(+) cells in atherosclerotic lesions are significantly higher, whereas the number of Mac2(+) and CD68(+) cells are lower in the ANGPTL4 treated atherosclerotic mice. However, numerous studies indicate that ANGPTL4 increases vascular inflammation and vascular permeability, ultimately promoting the development of atherogenesis [36]. In mesenteric lymph nodes, ANGPTL4 overexpression inhibited postprandial lipid uptake in macrophages, thus preventing macrophage transformation into foam cells and expression of inflammatory genes (CXCL2, CCR1, PTGS2, and GDF15) [11]. Silencing of ANGPTL4 in mice liver by antisense oligonucleotides decreases diet-induced obesity, dyslipidemia, glucose intolerance, and liver damage without generating severe safety issues [37,38].

As discussed above, the role and potential mechanisms of ANGPTL4 in inflammation appear to be elusive (Figure 1). Based on the literature, we conclude that the following aspects may partly explain the current conflicting function. Post-translational modifications play vital roles in ANGPTL4 regulation, such as glycosylation, phosphorylation, and myristoylation. Treatment with N-glycosidase resulted in a reduction in the molecular weight of fANGPTL4 demonstrating that ANGPTL4 is an N-glycosylated protein [39]. N-glycosidase also led to a significant decrease in the molecular mass of cANGPTL4, implying that cANGPTL4 contains complex oligosaccharide structures [40]. Sialylation also affects the pro- or anti-inflammatory effects of ANGPTL4. The distinct sialylation form of ANGPTL4 produced in podocytes could affect albuminuria and proteinuria. Supplementation with the sialic acid precursor confers renoprotection in diabetic nephropathy [41].

Abbreviations: FA: Fatty Acid; LPL: Lipoprotein Lipase; TG: Triglyceride.

Cleavage and oligomerization also exert crucial effects. ANGPTL4 undergoes post-translational cleavage by pro-protein convertases (PCs) at its RRKR-consensus cleavage site upon secretion, releasing n-terminal ANGPTL4 (nANGPTL4) and cANGPTL4. nANGPTL4 forms disulphide-linked dimers and tetramers. Oligomerization could increase the stability of nANGPTL4 and the ability to inhibit LPL [42]. Whether ANGPTL4 oligomerization impacts systemic inflammatory paradigms remains unclear. In addition, the correlation between truncated forms of ANGPTL4 and PC expression or activity remains unknown. During influenza pneumonia, the concomitant increase in furin activity cleaves fANGPTL4 to generate cANGPTL4, resulting in extensive lung injury characterized by host immune cell infiltration. cANGPTL4 immunoneutralization accelerates lung recovery significantly [16]. Recent findings indicated that nANGPTL4 inhibited metastasis while cANGPTL4 facilitated tumor metastasis revealed opposing functions of ANGPTL4/cANGPTL4 compared with nANGPTL4 in angiogenesis [43]. The changes of fANGPTL4/cANGPTL4 in inflammation may have different effects.

The function of ANGPTL4 is also regulated by its subcellular localization in specific cells and tissues. Data from the Human Protein Atlas suggests that ANGPTL4 is localized to the nucleoplasm and vesicles. Recent findings indicated that ANGPTL4 is enriched in exosomes [44]. More attention should be paid to the ANGPTL4’s secretory function and its role in nucleoplasm and cytoplasm. Several other factors are also probably involved. The effect of ANGPTL4 in the early and late stages of stomatitis could lead to different outcomes [45]. Current reports rarely find ANGPTL4 receptor presence in inflammatory diseases, the detailed mechanism of ANGPTL4 in inflammation needs to be explored in further studies. Moreover, it is reported that ANGPTL4 is silenced by aberrant DNA methylation of CpG islands during the development of human gastric cancers and carcinomas [46,47]. DNA methylation-mediated downregulation of ANGPTL4 promotes colorectal cancer metastasis by activating the ERK pathway [48]. Whether ANGPTL4 methylation plays a key role in inflammatory diseases requires further studies.

The present study has several limitations. First, it is unclear whether ANGPTL4 directly regulates inflammation or indirectly regulates this process by affecting lipid metabolism. Second, most of the literature focus on ANGPTL4 only and ignore other ANGPTLs with similar functions, such as ANGPTL3 and ANGPTL8. Third, the pathogenesis of ANGPTL4 has not yet been fully elucidated, but both intrinsic pathway and extrinsic pathway are assumed to play a role.

In summary, there is strong evidence that ANGPTL4 plays a dual role in the inflammatory response. Further studies are needed to elucidate the regulatory mechanisms, including the conditions that elevate ANGPTL4 expression in specific diseases, the extracellular or intracellular signaling pathways that mediate ANGPTL4 signaling, or how the outcome of this regulation affects only the local cellular microenvironment or leads to systemic inflammation.