text:
Endoglin aggravates peritoneal fibrosis by regulating the activation of TGF-β/ALK/Smads signaling

Background: Peritoneal fibrosis (PF) is an intractable complication in patients on long-term peritoneal dialysis (PD). Transforming growth factor-β (TGF-β) is a key pro-fibrogenic factor involved in PD-associated PF, and endoglin, as a coreceptor for TGF-β, plays a role in balancing the TGF-β signaling pathway. Here, we investigated whether endoglin could be a potential therapeutic target for PF. Methods: In vivo, we established PF model in SD rats by daily intraperitoneal injection of peritoneal dialysis fluids (PDF) containing 4.25% glucose for 6 weeks and downregulated endoglin expression by tail vein injection of AAV9-ENG on day 14 to assess the effect of endoglin on peritoneal morphology and markers related to fibrosis, angiogenesis, and epithelial-mesenchymal transition (EMT). In vitro, we treated human peritoneal mesothelial cells (HPMCs) transfected with ENG siRNA in high glucose medium to explore the potential mechanism of endoglin in PF. Results: Compared to control group, continuous exposure to biologically incompatible PDF induced exacerbated PF, accompanied by a significant increase in endoglin expression. Conversely, knockdown of endoglin ameliorated peritoneal injury characterized by increased peritoneal thickening and collagen deposition, angiogenesis, as well as EMT. Consistently, HPMCs cultured in high glucose medium underwent the EMT process and exhibited over-expression of fibronectin, collagen type I, vascular endothelial growth factor (VEGF), whereas these aforementioned alterations were alleviated after ENG siRNA transfection. In addition, we also found that ENG siRNA inhibited TGF-β-induced phosphorylation of Smad2/3 and Smad1/5/9 in HPMCs treated with high glucose (HG). Conclusion: Our findings confirmed for the first time that endoglin exacerbated PF by regulating the activation of TGF-β/ALK/Smads signaling, which will provide a novel potential therapeutic target in PF.

Introduction
Chronic kidney disease (CKD) has become a major public health problem in recent years due to the concomitant increase in the prevalence of its main associated risk factors, such as hypertension, diabetes and obesity (Lv and Zhang, 2019;Yang et al., 2020). As the disease continues to progress, the majority of CKD patients develop into end-stage renal disease (ESRD), a terminal stage requiring renal replacement treatment such as hemodialysis, peritoneal dialysis (PD), and kidney transplants. According to a recent report, clinical outcomes of PD are comparable to or better than hemodialysis, and PD patients can obtain a better quality of life (Li et al., 2017;Wong et al., 2018;Chuasuwan et al., 2020). However, PF limits the long-term application of PD in ESRD patients. Continuous exposure to bioincompatible, hypertonic PDF induces structural (fibrosis, angiogenesis, EMT) and functional (ultrafiltration failure) alterations in peritoneum, which eventually forces patients to withdraw from PD (de Lima et al., 2013;Zhou et al., 2016;Zhang et al., 2017;Balzer, 2020).
Mounting evidence indicates that PF pathophysiology involves fibrotic process itself, angiogenesis, and EMT (Balzer, 2020). The EMT process is a key mechanism contributing to PF. Mesothelial cells (MCs) that have undergone EMT can secrete pro-fibrogenic and angiogenic cytokines, inducing extracellular matrix (ECM) accumulation and vascularization (Selgas et al., 2006). The TGF-β signaling pathway is reported to play a predominant role in mediating PF, angiogenesis, and EMT. In HPMCs, prolonged exposure to PDF stimulates the production of TGF-β, which activates TGF-βRII and RI in turn. Subsequently, the TGF-β signaling pathway is transduced via phosphorylation of Smad2/3 and Smad1/5/8 and consecutive translocation of Smad4-Smad2/ 3 complex and Smad4-Smad1/5/8 into the nucleus. Various therapy strategies to attenuate PF by blocking the TGF-β/ ALK/Smads signaling pathway have been reported. TGF-β1 receptor inhibitor GW788388 effectively inhibited TGF-β1induced EMT in HPMCs, as well as improved PF and peritoneal membrane function in a PF mouse model (Lho et al., 2021). Similarly, blockade of thrombospondin-1 (TSP-1), a natural activator for TGF-β1, or siRNA-mediated knockdown of TGF-β both protected the peritoneal membrane from PDF-Induced damage (Yoshizawa et al., 2015;Jiang et al., 2019). Moreover, Smad3 knockout mice prevented PF and peritoneal dysfunction by attenuating EMT and reducing collagen formation (Duan et al., 2014).
Endoglin is a 180 kDa, homodimeric transmembrane glycoprotein that acts as an auxiliary receptor for ligands of the TGF-β superfamily. The TGF-β/endoglin/ALK/Smads signaling pathway has been reported to play a central role in several key cellular processes, including cell proliferation/ migration, EMT, ECM synthesis and angiogenesis, making endoglin a star molecule for targeting tumorigenesis (Kwon et al., 2017;Kasprzak and Adamek, 2018;Paauwe et al., 2018;Jeng et al., 2021). There are a growing number of clinical trials testing the combination of TRC105, a chimeric immunoglobulin G1 monoclonal antibody that binds endoglin, with standard therapies in different tumor types, showing that TRC105 is a promising strategy against solid tumors (Choueiri et al., 2019;Liu et al., 2020;Choueiri et al., 2021). Since fibrosis, angiogenesis and EMT are also involved in the initiation and progression of PF, we speculated that targeting endoglin might also have a therapeutic effect in PF.
Hence, in this study, we investigated the role and mechanism of endoglin in PF, which will provide the evidence that supports the therapeutic targeting of endoglin as a novel clinical strategy for intervention of PF.

Construction of AAV9-ENG
Recombinant adeno-associated virus (AAV9-ENG) and adeno-associated virus negative control (AAV9-NC) were constructed by Genomeditech (Shanghai, China). The single-stranded DNA oligo containing the interfering sequence was first synthesised, then annealed and paired to produce a double-stranded DNA oligo, which was then directly ligated into the enzymatically cleaved RNAinterfering adeno-associated viral vector through the enzymatic cleavage sites at both ends. The constructed adeno-associated virus vector and its helper packaging plasmid were co-transfected into AAV-293 cells. After 6-8 h of transfection, the medium was replaced with fresh medium and the Enhancing buffer was added. The cells were continued to be cultured for 72 h. The virus-rich cells and supernatant were collected after cell detachment, concentrated and purified to obtain a high concentration of virus concentrate. The viral titer was 1.10 × 10^13 V g/ml determined using quantitative PCR. The rat peritoneum was taken 4 weeks after AAV injection to evaluate the transfection effect by Western blot and immunohistochemical staining. In addition, since our adeno-associated viral vector carries the EGFP gene, its expression product fluoresces green under fluorescent microscopy. Therefore, fluorescence analyses of frozen sections can also be used to verify the ability of AAV9 vector to transduce peritoneum.

Animal and experiments
The study protocol was approved by the principles of the Animal Care Ethics Committee of Shandong Provincial Hospital Affiliated with Shandong University. 32 male SD rats (6-8 weeks old), provided by Jinan Pengyue Experimental Animal Breeding Co., (Jinan, Shandong), were randomly divided into four groups (n = 8/group): Control group, PDF group, AAV9-NC+PDF group, AAV9-ENG+PDF group. To successfully establish PF animal model, we treated rats in PDF group with daily intraperitoneal injection of 4.25% glucose PDF (Baxter Healthcare Ltd., Deerfield, IL, United States) for 6 weeks. Control rats received an equal volume of normal saline (NS). Two weeks after the start of the experiment, AAV9-ENG (The viral titer was 1.10 × 10^13 V g/ml, 100 μl per rat), constructed by Genomeditech (Shanghai, China), was given to rats in the AAV9-ENG+PDF group by tail vein injection, while AAV9-NC was applied as a control to rats in the AAV9-NC group. Four weeks after AAV injection, we harvested anterior abdominal wall and fixed in 4% paraformaldehyde for pathological analysis, as well as stored omentum tissue under −80°C for protein expression analysis.

Histopathologic evaluation
The peritoneum samples fixed in 4% paraformaldehyde underwent a series of standard procedures including successive dehydration in a graded alcohol series (75%, 85%, 95%, and 100%, v/v), transparency in dimethylbenzene and paraffin embedding, and then were sectioned into 5 µm thick slices. For histological examination, hematoxylin-eosin (HE) and Masson's trichrome staining were conducted to show the pathological changes and peritoneum thickness according to standard protocols provided by the manufacturer (Sigma-Aldrich). We utilized ImageJ (National Institute of Health, Bethesda, MD), a computerized image analysis software, to evaluate PF by measuring the percentage of peritoneal membrane occupied by collagen fibers. In addition, peritoneum thickness was assessed by measuring the distance from the surface mesothelium to the upper limit of the muscular tissue with the aid of ImageJ software.

Immunohistochemical investigation
The immunohistochemistry staining was performed to assess protein expression using the streptavidin-peroxidase immunohistochemical method. Paraffin-embedded tissue sections were dewaxed, hydrated and heat-treated with antigen recovery buffer (PH = 6). Subsequently, the sections were subjected to 3% hydrogen peroxide at room temperature for 10 min, then blocked with normal goat serum at room temperature for 15 min and incubated overnight at 4°C with primary antibodies as follows: Rabbit anti-endoglin (Proteintech, 10862-1-AP, 1:1000), rabbit anti-fibronectin (Proteintech, 15613-1-AP, 1:500), rabbit anti-collagen type I (Proteintech, 14695-1-AP, 1: 500) and rabbit anti-CD31 (Abcam, ab182981, 1:1000). On the following day, the sections were washed with PBS and incubated with HRP-conjugated goat anti-rabbit IgG secondary antibodies for 15 min at room temperature, followed by incubation for 15 min with horseradish peroxidase-labeled streptavidin. After incubation for 5-10 min with DAB, the sections were counterstained with hematoxylin. Finally, the selected regions were captured under microscope (Leica, Germany).

siRNA transfection
We used siRNA targeting ENG (Genomeditech, Shanghai, China) to further identify the role of endoglin in PF. The target sequences were listed as follows: ENG siRNA: 5′-CAAUGAGGCGGUGGCAAU-3′; NC siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′. According to the manufacturer's standard protocol, ENG siRNA and NC siRNA were transiently transfected into cultured HPMCs with 60%-80% of confluence using Lipofectamine 2000 (Invitrogen, CA, United States). Then transfected cells were treated with high glucose for subsequent experiments.

Cell counting kit-8 assay
The Cell Counting Kit-8 (CCK-8) (Elabscience, Wuhan, China) was used to detect the effect of different concentrations of HG on the proliferation of HPMCs. We seeded HPMCs in 96-well culture plates at a density of Frontiers in Pharmacology frontiersin.org 03 3,000 cells per well. When the cells reached confluence, they were cultured in serum-free medium overnight under starvation conditions. We then treated them with different concentrations of HG (control, 1.5%, 2.5%, or 4.25%). After 48 h, 90 µl of fresh serum-free medium and 10 µl of CCK-8 were added to each well, followed by incubation for 2 h at 37°C. Finally, we measured the absorbance of each well at 450 nm with a microplate reader (EL340 BioTek Instruments, Hopkinton, MA, United States).

Statistical analysis
All experiments in this research were independently repeated at least three times. The data processed by GraphPad Prism 8.0.2 were presented as the mean ± SD. We evaluated statistical significance between two groups using Student's t-test. To compare the difference among multiple groups, one-way ANOVA was used. p < 0.05 was considered as statistically significant.

Endoglin was upregulated in peritoneal fibrosis rats
To determine the relationship between endoglin and PF, we firstly constructed PD-related PF model by daily intraperitoneal injection with 4.25% dextrose PDF. H&E staining and Masson trichrome staining revealed that, compared with control group, there was a significant increase in peritoneal membrane thickness and collagen fibrils accumulation in the PDF group ( Figure 1A). The western blot results also showed increased expression of fibronectin and collagen type I in the PDF group ( Figures 1B,C). We then detected the protein expression level of endoglin in the peritoneal anterior abdominal wall and omentum from rats treated with PDF. The immunohistochemistry staining and western blot assays indicated that the endoglin expression in PDF rats is higher than that in control group ( Figures 1B-D). In general, we confirmed that endoglin was increased in rats with PD-related PF.

Knockdown of endoglin attenuated peritoneal dialysis-induced peritoneal fibrosis
To elucidate the role of endoglin in PF, we knocked down endoglin by injecting AAV9-ENG into tail vein of rats. The western blot results and immunohistochemical study showed that AAV9-ENG efficiently transduced into rat peritoneum and downregulated the increase of peritoneal endoglin expression induced by PDF (Figures 2A,B). We then evaluated whether endoglin deficiency would alleviate PD-induced PF. The results obtained from H&E staining, Masson trichrome staining and fluorescent images of frozen peritoneum sections exhibited improved peritoneal thickness and less collagen fiber deposition in AAV9-ENG+PDF group ( Figures 2C,D). In addition, western blot results and immunohistochemical Frontiers in Pharmacology frontiersin.org 04

FIGURE 1
Endoglin was upregulated in PF rats. (A) Representative images of HE and Masson's trichrome staining in peritoneum in control and PDF rats. Scale bar = 100 μm. Mean peritoneal membrane thickness and peritoneal fibrosis score in control and PDF rats. (B) Fibronectin, Collagen Type I and endoglin protein levels in control and PDF rats. (C) The graphs showed the quantitative analysis of Fibronectin, Collagen Type I and endoglin protein levels. (D) Representative immunohistochemical staining images of endoglin in control and PDF rats. The blue arrows indicate vascular endothelial cells. Scale bar = 100 μm. n = 6. All quantitative data are expressed as mean ± SD, *p < 0.05, **p < 0.01.

Frontiers in Pharmacology
frontiersin.org 06 staining suggested that endoglin knockdown decreased the expression levels of fibrotic markers fibronectin, collagen type I ( Figures 2D,E). Taken together, these results indicated that endoglin was involved in PF progression.

Angiogenesis in endoglin knockdown rat
Since peritoneal angiogenesis is proven to play a key role in the development of PF, we explored whether endoglin deficiency could ameliorate angiogenesis by immunohistochemical staining for CD31, a marker of capillary sprouting. As shown in Figure 3A, exposure to PDF increased the number of CD31-positive vessels in the rat peritoneum, and the AAV9-ENG treatment inhibited this response. Moreover, considering the involvement of VEGF in peritoneal angiogenesis, we conducted western blot analysis for its expression. Compared with control, we observed a notably increased VEGF in PDF rats, which could be reversed by endoglin knockdown ( Figure 3B). Together, these data demonstrated that endoglin deficiency could ameliorate peritoneal angiogenesis.

Epithelial-mesenchymal transition in endoglin knockdown rat
Given the critical role of EMT in PF, we evaluated the protein expression levels of EMT biomarkers E-cadherin, N-cadherin, α-SMA, and vimentin by western blot analysis to further investigate the impact of endoglin on EMT in PDF rats. We observed that PDF treatment induced downregulation of E-cadherin and upregulation of N-cadherin, α-SMA and vimentin compared with the control group, while endoglin deficiency reversed the above changes ( Figures 4A,B). These results suggested that endoglin knockdown could reverse EMT process, which in turn acts to alleviate PF.

Endoglin was upregulated in high glucose stimulated human peritoneal mesothelial cells
To further identify the role of endoglin in PD-associated PF, we stimulated HPMCs with various concentrations of glucose (control, 1.5%, 2.5%, 4.25%) for 24 h. First, we assessed the

Frontiers in Pharmacology
frontiersin.org impact of HG on cell proliferation by performing CCK-8 experiments and chose 2.5% glucose for subsequent experiments based on the results ( Figure 5A). Then, we found that HG resulted in injury to HPMCs consistent with in vivo experiments, as evidenced by the downregulation of E-cadherin protein expression and the increased protein expression levels of N-cadherin, α-SMA, vimentin, fibronectin, collagen type I and VEGF ( Figures  5B,C). By western blot analysis, we further detected endoglin expression and proved a significant increase of endoglin in HG-induced HPMCs ( Figures 5B,C). Collectively, these results confirmed that increased endoglin protein level parallel changes in the expression levels of fibrosis, angiogenesis, and EMT-related proteins in HG-induced HPMCs.

Downregulation of endoglin improved high glucose-induced injury in human peritoneal mesothelial cells
In order to further determine whether endoglin is involved in HG-induced HPMCs injury in vitro, we transfected ENG siRNA into HPMCs treated with HG to silence endoglin expression. Western blot results showed that endoglin deficiency resulted in a dramatic increase in E-cadherin protein levels and a significant decrease in N-cadherin, α-SMA, vimentin, fibronectin, collagen type I and VEGF protein expression levels in the HPMCs treated with 2.5% glucose alone ( Figure 6A). The transfection efficiency of ENG siRNA was verified by western blot analysis ( Figure 6B). Besides, Immunofluorescence staining also illustrated that ENG siRNA led to a notably increase of epithelial marker E-cadherin but decrease of mesenchymal marker α-SMA in the HPMCs after HG (2.5%) stimulation ( Figure 6C).

Endoglin deficiency inhibited the transforming growth factor-β/ALK/smads pathway involved in high glucose-induced human peritoneal mesothelial cells injury
Given the role of endoglin as TGF-β co-receptor, we performed western blot assay to elucidate whether endoglin mediated HG-induced HPMCs injury by the involvement of the TGF-β/ALK/Smads signaling pathway. Compared with control, HG (2.5%) treatment significantly elevated the protein expression level of TGF-β1 and increased the phosphorylation levels of Smad2, Smad3 and Smad1/5/9, indicating that the TGF-β/ALK/Smads pathway was activated in HG-induced HPMCs injury ( Figures 7A,B). We then tested the protein levels of TGF-β1, pSmad2, pSmad3 and pSmad1/5/9 in endoglin knockdown HPMCs stimulated with HG (2.5%) and found that ENG siRNA inhibited the expression and phosphorylation levels of these factors in HG group (Figures 7A,B). Together, these data revealed that endoglin deficiency inhibited activation of the TGF-β/ ALK/Smads pathway in HG-induced HPMCs injury. The graphs showed the quantitative analysis of E-cadherin, N-cadherin, α-SMA and vimentin protein levels. n = 6. All quantitative data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Frontiers in Pharmacology
frontiersin.org

Discussion
PF is an unavoidable complication concomitant with longterm PD and is associated with poor prognosis in patients with ESRD. Based on the existing studies on the mechanism of PF, it can be summarized as a pathological process containing the crosstalk of fibrosis, angiogenesis and EMT (Balzer, 2020). TGFβ, a key fibrogenic factor, mediates fibrogenesis in PF. Endoglin acts as a co-receptor for TGF-β, exerting its effect by modulating TGF-β signal transduction. In this study, we evidenced that endoglin positively correlates with the development of PF by conducting in vivo and in vitro experiments. Moreover, our findings in the present study indicated that the HG/TGF-β/ endoglin/ALK/Smads signaling pathway exerted an import influence on the pathogenesis of PF.
In recent years, mounting evidence indicates that endoglin is closely related to organ fibrosis. Several studies have identified that endoglin plays a pivotal role in cardiac fibrosis of different heart failure models and targeting endoglin attenuates cardiac fibrosis (Kapur et al., 2012;Lin et al., 2016;Shyu, 2017); Endoglin has also been found to be upregulated in renal biopsy samples from patients with diabetic nephropathy and in renal fibrosis models, including unilateral ureteral obstruction (UUO) and radiation-induced nephropathy (Rodriguez-Pena et al., 2002;FIGURE 5 Endoglin was upregulated in HG stimulated HPMCs. (A) HPMCs were treated with different concentrations of HG (control, 1.5%, 2.5%, 4.25%) for 24 h, and CCK8 were performed to detect the viability of HPMCs. (Data are shown as mean ± SD, ***p < 0.001 compared with the control, n = 3). (B) Western blot detection of endoglin, Fibronectin, Collagen Type I, E-cadherin, N-cadherin, vimentin, α-SMA and VEGFA of peritoneum in different groups. GAPDH was used as a loading control. The results from the quantitative analysis are shown in (C). (Data are shown as mean ± SD, *p < 0.05 compared with the control, **p < 0.01 compared with the control, ***p < 0.001 compared with the control, ****p < 0.0001 compared with the control).
Frontiers in Pharmacology frontiersin.org 09 Scharpfenecker et al., 2009;Munoz-Felix et al., 2014). However, to our knowledge, there are no studies on the role of endoglin in PF. In this study, we simulated PD process by daily intraperitoneal injection of PDF containing 4.25% HG, and found that endoglin was upregulated in the rat peritoneum treated with PDF. We also proved that HG injury induced fibrosis of HMPCs, accompanied by elevated expression of endoglin. To further validate our hypothesis, we Frontiers in Pharmacology frontiersin.org 10
Frontiers in Pharmacology frontiersin.org 11 downregulated endoglin expression both in vivo and in vitro and discovered that inhibition of endoglin partially reversed PF, suggesting that endoglin-targeting therapy may be effective in the treatment of PF.
It has been known that angiogenesis is a key event in the development of PF. Williams et al. (2002) performed the first comprehensive cross-sectional analysis of the morphological changes in the peritoneal membranes of patients undergoing PD and showed that the density of blood vessels per unit length of peritoneum was significantly higher for patients with peritoneal membrane function failure and was correlated with the degree of fibrosis. Furthermore, Zhu et al. (2021) proved that Tetramethylpyrazine (TMP) treatment inhibited peritoneal angiogenesis and also ameliorated peritoneal thickening and collagen deposition. These findings suggest that antiangiogenic therapy may serve as a potential modality for preventing or reversing PF. Among all molecules that regulate peritoneal angiogenesis, VEGF as a strong pro-angiogenic factor binds to VEGFR expressed on the cell surface of vascular endothelial cells, stimulating the formation of new capillaries in the peritoneal membrane. Increased effective vascular surface area accelerates glucose uptake from the dialysate into the circulation, leading to a dissipation of glucose-driven osmotic pressure, which ultimately results in ultrafiltration failure (UF) (Aroeira et al., 2005;Saxena, 2008;de Lima et al., 2013;Shi et al., 2022). The selective high expression of endoglin on tumour vessels has led to widespread attention to it as a target for cancer therapy, reminding us whether anti-endoglin treatment could similarly improve peritoneal angiogenesis and thus reduce PF (Paauwe et al., 2016;Kasprzak and Adamek, 2018;Liu et al., 2020). In this study, we found that treatment with 4.25% dextrose PDF significantly triggers peritoneal angiogenesis as evidenced by the increased level of CD31, an endothelial cell marker, and VEGF. However, anti-endoglin therapy inhibited peritoneal angiogenesis. We also found that non-epithelioid mesothelial cells (MCs) secreted more VEGF than epithelioid MCs, which is consistent with previous reports that VEGF is expressed abundantly in PMCs, particularly in PMCs undergoing the EMT process (Selgas et al., 2006;Aroeira et al., 2007;Lopez-Cabrera, 2014). In contrast, endoglin deficiency decreased production of VEGF in HPMCs. Obviously, just as previous studies have shown an interconnection between endoglin and the VEGF signaling pathway, we speculate that the same is true for peritoneal angiogenesis during PD (Li et al., 2015;Jadhao et al., 2022). Although the relationship in detail needs to further elucidate, it is sure that endoglin may also be a therapeutic target for the treatment of peritoneal angiogenesis and UF.
EMT is a dynamic process during which epithelial cells acquire mesenchymal phenotypes and behavior following the downregulation of epithelial features (Lamouille et al., 2014;Singh et al., 2018;Dongre and Weinberg, 2019;Sha et al., 2019). As mentioned above, converging lines of evidence suggest that angiogenesis and fibrosis are closely interconnected through the EMT process, which makes it become a key mechanism to intervene the progression of PF. Endoglin has been proved to be involved in the development of cardiac valve and in the cell migration and invasion in endometriosis, ovarian cancer and clear cell renal carcinoma, all of which are achieved through regulation of the EMT process (Mercado-Pimentel et al., 2007;Hu et al., 2019;Chen et al., 2021;Zhang et al., 2021). In this study, we discovered that loss of endoglin expression mediated by either AAV9-ENG or ENG siRNA caused the reversal of EMT process and corresponding changes in the protein expression level of EMT markers including E-cadherin, N-cadherin, vimentin and α-SMA. Our findings suggest that endoglin may aggravates PF through modulation of EMT.
Among the molecular pathways involved in the PF, the TGFβ signaling pathway, comprising Smad-dependent and Smadindependent signaling, is one of the most hotly discussed (Balzer, 2020). In this study, we mainly focus on the canonical Smad signaling pathway that dominates in PF. This signaling pathway is initiated by binding of TGF-β or BMP ligands to TGF-β RII, which subsequently causes recruitment, transphosphorylation and activation of TGF-β RI (ALK5 or ALK1) and finally phosphorylates downstream Smad2/3 or Smad1/5/8, leading to signal transduction (Pomeraniec et al., 2015;Seystahl et al., 2015;Liu et al., 2020;Schoonderwoerd et al., 2020;Burghardt et al., 2021;Gonzalez Munoz et al., 2021). On the one hand, when the peritoneal membrane is continuously exposed to bioincompatible PDF, it is certain that the TGF-β1/ALK5/ Smad2/3 signaling pathway is significantly activated in PMCs. In this study, we also demonstrated its activation in HGstimulated HPMCs, as evidenced by the upregulation of TGF-β1 and phosphorylated Smad2 and Smad3 levels. At present, there exists controversy over whether the ALK1/Smad1/5/ 8 signaling pathway is activated. Loureiro et al. (2010) considered that MCs constitutively express BMP-7 and exhibit basal activation of BMP7-dependent Smad1/5/8 in vitro, and that these are downregulated in the presence of conventional PDF (Phillips and Fraser, 2010). In contrast, Zhang et al. (2020) demonstrated that TGF-β1 induced significant activation of the Smad1/5/9 pathway in HMrSV5 cells. In our study, the phosphorylation level of Smad1/5/9 was upregulated in HGinduced HPMCs. In conjunction with the account in studies related to organ fibrosis that the Smad1/5/9 signaling activity tends to be opposite to the pro-fibrotic, EMT-promoting activity possessed by the Smad2/3 signaling pathway, we suggest that activation of the Smad1/5/9 signaling pathway may be the result of antagonism of the Smad2/3 signaling pathway activation. Overall, the epithelial/mesenchymal status of PMCs is determined by the balance between Smad2/3 and Smad1/5/ 9 signaling and the Smad2/3 pathway favors the mesenchymal phenotype (Strippoli et al., 2016). We speculate that the level of phosphorylated smad1/5/9 measured by immunoblotting may be influenced by the time of stimulation, showing an increase, Frontiers in Pharmacology frontiersin.org decrease or even no change, which requires further study. On the other hand, endothelial cells (ECs) undergo proliferation and migration to promote angiogenesis, which is seen as another essential step in the development of PF. Numerous studies have shown that TGF-β can activate both the ALK5 and ALK1 pathways in ECs. The activation of ALK1/Smad1/5/ 8 pathway promotes the proliferation and migration of ECs and thus exerts a pro-angiogenic effect, whereas the opposite is true for ALK5/Smad2/3 signaling pathway. Considering that TGF-β signaling pathway in ECs during angiogenesis has been reported in great detail, we did not further explore the ALK5 and ALK1 pathways in ECs exposed to conventional PDF in the present study.
Given the important role of balance between the Smad2/3 and Smad1/5/8 pathways mediated by TGF-β in the development of PF and the known role of endoglin in regulating TGF-β signaling, we explored how endoglin regulates TGF-β signaling in PMCs exposed to HG and thus exerts an inductive effect on PF in this study. Endoglin promotes TGF-β-induced Smad2/3 or Smad1/5/9 responses by interacting with ALK5 or ALK1 to form a TGF-β receptor signaling complex. Notably, the effect of endoglin on TGF-β signaling depends on the type of cell and tissue as well as the cellular context. In primary chondrocytes, endoglin enhances the ALK1/Smad1/5/8 pathway associated with catabolism and inhibits the ALK5/Smad2/3 pathway associated with anabolism, whereas endoglin may mediate cartilage formation via the ALK5/Smad2/3 pathway in mesenchymal stem cells (MSCs) expressing endoglin (Finnson et al., 2010;Bianchi et al., 2021). High endoglin expression stimulates the ALK1 pathway and indirectly inhibits ALK5 signaling, thus promoting the activation state of angiogenesis. In contrast, in the absence of endoglin, the TGF-β/ALK5 signaling pathway predominates and maintains quiescent the endothelium (Lebrin et al., 2004;Lebrin et al., 2005;Ollauri-Ibanez et al., 2017). In hepatocellular carcinogenesis (HCC), endoglin promotes ECs proliferation, migration and angiogenesis through activation of the ALK1/Smad1/5/8 pathway. Conversely, endoglin increases extracellular matrix (ECM) synthesis via the enhancement of ALK5/smad2/3 signal transduction (Jeng et al., 2021). In addition to the examples of differential regulation of ALK1/Smad1/5 and ALK5/Smad2/3 by endoglin already discussed above, O'Connor et al. (2007) reported that endoglin acts as a positive regulator of both ALK1-induced Smad1/5/8 activation and ALK5-induced Smad2/3 activation in bone marrow stromal cells. In the present study, we similarly found that siRNA-mediated endoglin knockdown inhibited HG-induced phosphorylation of smad2/3 and Smad1/5/9 in HPMCs, suggesting that endoglin is involved in HG-induced HPMCs injury through the canonical TGF-β pathway. The only plausible explanation for conflicting data is that there are two different alternatively spliced isoforms of endoglin, L-endoglin (L, long) and S-endoglin (S, short), which differentially modulate TGF-β signaling mediated by ALK1 and ALK5 (Perez-Gomez et al., 2005;Velasco et al., 2008;Blanco et al., 2015). L-endoglin enhanced the ALK1 pathway, while S-endoglin promoted the ALK5 pathway.
Although some researchers have investigated the role of endoglin by generating stable transfectants of U937 cells or L6E9 myoblasts to overexpress the L-or S-endoglin isoforms, appropriate tools to clearly distinguish between them are still lacking as of today. Therefore, most studies published on endoglin barely have made little distinction between L-and S-endoglin. In the present study, we found that both the ALK1/Smad1/5/9 and ALK5/Smad2/3 signaling pathways were activated by endoglin, but did not further distinguish between L-endoglin and S-endoglin and investigate their respective effects on TGF-β signaling, which will be involved in our future studies.
In conclusion, we demonstrated that endoglin promotes PF, and downregulation of endoglin can relieve PF by suppressing EMT, fibrosis and angiogenesis. In addition, endoglin mediates peritoneal injury through canonical the TGF-β signaling pathway. Therefore, our findings suggest that targeting endoglin may be a therapeutic intervention to preserve the integrity of peritoneum for long-term PD patients.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the Animal Care Ethics Committee of Shandong Provincial Hospital Affiliated with Shandong University.


Role of fatty acid transport protein 4 in metabolic tissues: insights into obesity and fatty liver disease

Abstract Fatty acid (FA) metabolism is a series of processes that provide structural substances, signalling molecules and energy. Ample evidence has shown that FA uptake is mediated by plasma membrane transporters including FA transport proteins (FATPs), caveolin-1, fatty-acid translocase (FAT)/CD36, and fatty-acid binding proteins. Unlike other FA transporters, the functions of FATPs have been controversial because they contain both motifs of FA transport and fatty acyl-CoA synthetase (ACS). The widely distributed FATP4 is not a direct FA transporter but plays a predominant function as an ACS. FATP4 deficiency causes ichthyosis premature syndrome in mice and humans associated with suppression of polar lipids but an increase in neutral lipids including triglycerides (TGs). Such a shift has been extensively characterized in enterocyte-, hepatocyte-, and adipocyte-specific Fatp4-deficient mice. The mutants under obese and non-obese fatty livers induced by different diets persistently show an increase in blood non-esterified free fatty acids and glycerol indicating the lipolysis of TGs. This review also focuses on FATP4 role on regulatory networks and factors that modulate FATP4 expression in metabolic tissues including intestine, liver, muscle, and adipose tissues. Metabolic disorders especially regarding blood lipids by FATP4 deficiency in different cell types are herein discussed. Our results may be applicable to not only patients with FATP4 mutations but also represent a model of dysregulated lipid homeostasis, thus providing mechanistic insights into obesity and development of fatty liver disease.

Introduction
Dietary fats, especially fatty acids (FAs), are essential biochemicals for cellular structures and energy production. In organisms, FAs essentially participate in all cellular metabolic pathways with the requirement that they first are activated to their acyl-CoA derivatives. Acyl-CoAs participate in three main metabolic pathways including β-oxidation to generate energy, esterification to be stored as triacylglycerols/triglycerides (TGs), and the synthesis of structural lipids, such as phospholipids (PLs), sphingolipids (SPLs), plamalogens, and cholesterol esters (CEs). Other pathways include FA elongation, insertion and removal of double bonds, protein acylation, gene expression, signalling, and regulation of intracellular functions. The physiological and nutritional states dictate which direction FAs are trafficked towards [1][2][3]. The gut is the organ designated for FA absorption [4]. The liver is the most important organ for regulation of FA metabolism [5,6]. The muscle and adipose tissues predominantly play a role in FA oxidation and storage [7][8][9][10][11]. The state of homeostatic FA metabolism in these tissues and blood circulation can be seen by clinical biochemistry parameters, such as, blood lipids [12], liver fats [13,14], insulin release [15], and the state of insulin sensitivity [16][17][18]. Recently, metabolic (dysfunction) associated fatty liver disease (MAFLD) has been suggested by experts as a more appropriate terminology than the commonly used non-alcoholic fatty liver disease (NAFLD) [19][20][21][22], thus emphasizing the importance of metabolic homeostasis. In this context, the disturbance in FA metabolic homeostasis is known to contribute to MAFLD/NAFLD, obesity, and insulin resistance [23,24].
FATP4 is localized mainly at the endoplasmic reticulum (ER) and partially localized to peroxisomes [55,60]. It plays a role in FA transport via ACS-activated vectorial acylation [48,55,[60][61][62][63], that the depleted intracellular FA concentrations indirectly trigger an FA uptake. Metabolic labelling ACS activity assays show that FATP4 overexpression increases the activity by 2 folds with palmitate and 5 folds with lignocerate indicating that the FATP4 gene encodes an ACS with substrate specificity towards VLCFAs [62,63]. Remarkably, FATP4 mutations or deletion cause ichthyosis premature syndrome (IPS) and lethality of neonates in humans [74,75] and mice [76][77][78][79]. An uptake/flux study using 14 C-radiolabeled oleic acid has demonstrated that FATP5 or ACSVL6 which is localized on the plasma membrane, functions as an FA transporter in the hepatocytes [80]. Consistently, FATP5 silencing in vivo attenuates diet-induced NAFLD [81]. Lastly, FATP6 or ACSVL6 is shown to be an FA transporter and is reported only in human heart [40]. Taken together, FATP enzymes play a povital role in FA uptake and metabolism based on their diverse expression and cellular localization in different cell types. Thus, altered expression of FATPs in metabolic tissues have been recognized to play a major role in diet-induced obesity, hepatic steatosis, insulin resistance, and metabolic syndrome diseases in mice [64,65,68,72,81] and humans [23,24,38,82,83].
In the overall lipid metabolism, ACS or FATP enzymes also play a significant role in FA degradation pathway via αand β-oxidation. It is generally accepted that β-oxidation of LCFAs and VLCFAs occurs primarily in mitochondria and peroxisomes, respectively. By using [ 14 C]oleate in β-oxidation metabolic labelling assay, it is demonstrated that hepatic lon-chain fatty ACS1 (ACSL1) is important for mitochondrial β-oxidation of LCFAs [84], Moreover, ACSL1-mediated β-oxidation in adipocytes is required in cold thermogenesis [85]. It is also reported that ACSL3 mediates β-oxidation of FAs in lung cancer cells [86]. For FATP family, FATP1, which is localized in mitochondria of mouse skeletal muscle, is shown to increase β-oxidation resulting in hyperketonemia in vivo [87]. Interests have been especially paid on β-oxidation of VLCFAs in peroxisomes and the identification of the responsible ACS/FATP enzymes. This is because the impairment of VLCFA degradation leads to abnormal accumulation of VLCFAs in tissues and plasma in patients with the neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) [56,88]. So far, ACSL4, FATP2, and FATP4 have been identified to be present in peroxisomes [56]. ACSL4 may have a specialized role in peroxisomes since it has specificity for activation of polyunsaturated fatty acids (PUFAs). While FATP2 activates both palmitate and lignocerate [57], this enzyme is a major contributor to peroxisomal ACSVL activity [72]. However, FATP2 deletion in mice does not lead to accumulation of VLCFAs in livers and kidneys, although VLCFA β-oxidation rates are reduced [88]. Likewise, fibroblasts from X-ALD patients show decreased perosixomal ACSVL activity; however, FATP2 expression is not significantly different between normal controls and X-ALD [89]. Thus, it appears that FATP2 does not have a direct role in X-ALD pathology. Metabolic labelling study on β-oxidation by the conversion of [1-14 C]FA to 14 CO 2 has demonstrated the specificity of FATP4 for VLCFA lignocerate [55]. Consistent with VLCFA preference and localization to peroxisomes, it has been shown that FATP4 interacts with fatty acid synthetase and the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP) whereby its mutations are the primary cause of X-ALD [90]. Mutations of ALDP however does not alter its interaction with FATP4. It is postulated that FATP4 localized at the cytoplasmic side of the peroxisomal membrane activates VLCFAs to CoAs which are transported by ALDP for β-oxidation in the peroxisomal lumen. Furthermore, it is shown that VLCFA-CoAs catalyzed by FATP4 can undergo elongation and these ultraVLCFA-CoAs are subsequently incorporated into complex lipids [91]. Thus, FATP4 paticipates in X-ALD pathology by elevating the levels of lipids containing VLCFAs and ultraVLCFAs in the cytosol which are exported extracellularly.
Among human FATP family members, FATP4 is broadly distributed and highly expressed in metabolically active tissues such as adipocytes [23,31,61,67,69] and adipose tissues of obese individuals [38,82,83]. Although the protein level of FATP4 is high in thyroid glands (Figure 1), there has been no investigations on FATP4 in these tissues. FATP4 gene is evolutionally conserved and is the only FATP known so far to cause severe neonatal abnormalities or death as reported in FATP4-null mice [76][77][78][79], as well as IPS patients with FATP4 mutations seen mainly in Europe [74,75,[92][93][94][95][96] and some in Asia [97,98]. The deletion of Fatp4 in mice or mutations of FATP4 in humans lead to wrinkle-free restrictive dermopathy with phenotypes including ichthyosis, embryo/neonatal lethality, and death shortly after birth. Interestingly, a targeted disruption in the upstream of exon 2 of Fatp4 gene [78] results in neonatal death at earlier time than the disruption of exon 3 [76]. This indicates a different truncated form of Fatp4 in mice. Furthermore, FATP4 deletion in mice leads to epidermal hyperproliferation [99], and these mutants also represent a model of congenital ichthyosis [100]. Of note, epidermis-specific inactivation of Fatp4 in mice does not lead to death but causes milder hyperkeratosis when compared with the phenotypes of Fatp4-null mice [99]. Speculatively, the severe phenotypes seen in Fatp4-null mice could be due to the deficiency in immune cells such as macrophages that might lead to inflammatory activation. In support of this notion, many patients with FATP4 mutations survive neonatal abnormalities, and adult patients show signs of not only hyperproliferative hyperkeratosis but also allergies and eosinophilia [92,101]. It is surmised that these patients may exhibit high blood lipids and insulin resistance [38,82,83] due to the mutations in metabolically active tissues, such as, adipose tissues and liver. In the past two decades, research efforts have been made to investigate FATP4 role in the skin of mice and humans. Key findings on Fatp4-null mice include a decrease in ceramides containing ultraVLCFAs [76,79,102] including omega-O-acylceramides [79,102] and omega-hydroxyceramides [102]. The latter two constitute the major compounds of the corneocyte lipid envelope which is required for normal permeability barrier in skin. Concomitant with decreased ultraVLCFA-ceramides, there is an accumulation of neutral lipids as reported in skin of IPS patients [75] and Fatp4-null mice [76,77]. As discussed in this review, more recent studies in our laboratories using tissue-specific knockout (KO) mice have suggested FATP4 functions on FA metabolism in metabolic tissues including the intestine, liver, and adipose tissues. We also have reviewed other published studies to discuss the relationships between FATP4 with obesity and fatty liver disease.

General characteristics of FATP4
Herrmann and co-workers [63] have stated that the murine Fatp4 gene contains 1929 bp in the open-reading frame encoding a polypeptide of 643 amino acids, and is assigned to mouse chromosome 2 band B (syntenic to the region 9q34 encompassing the human gene). In 2004, Gertow and co-workers [38] have stated that the human FATP4 gene contains 12-coding exons spanning more than 17 kb of genomic DNA and maps to chromosome 9q34. In 2007, Watkins and co-workers [35] have shown that the human FATP4 gene contains 13 exons and mapped the FATP4 gene to the plus strand of chromosome 9q34.11 by genomic sequence analysis ( Figure 2).
The FATP family is conserved from invertebrates to vertebrates [35]. FATP1 and FATP4 in vertebrates are clustered with FATP genes in invertebrates. FATP2, FATP3, FATP5, and FATP6 are present only in vertebrates among which FATP6 being present only in humans [103]. FATP1 and FATP4 are broadly distributed, and they could similarly exhibit the evolutionary conserved functions [103]. Within the coding region, the exon-intron structures of the murine Fatp4 gene are identical to its human counterparts and human FATP1 gene is closely resembled to the mouse gene [63]. Thus, FATP1 and FATP4 may play a complementary role because it has been shown that Fatp1 can compensate for Fatp4 in the developing mouse epidermis [104][105][106]. It is known that FATP1 is highly expressed in hormone-sensitive tissues including white adipose tissue and soleus muscle, and that a hormone insulin is necessary for FATP1 to mediate FA uptake [64,65]. Being different from FATP1, FATP4 indirectly mediates FA uptake in not only insulin-sensitive [61] but also insulin-insensitive tissues [55,60,62,63]. In adipocytes, the knockdown of FATP1 or FATP4 could decrease cellular TG levels by inducing TG lipolysis [67]. This knockdown also leads to an increase in the uptake of 2-deoxyglucose. As glucose is converted to acetyl-CoA and used for syntheses of fatty acids and subsequently TGs, hence FATP1 or FATP4 knockdown may replenish the decreased TGs by enhancing glucose uptake. It is thought that FATP1 and FATP4 may have overlapping biological functions, and that the increased FATP1 expression may reduce atopic abnormalities and death in surviving patients with FATP4 mutations [106].
In humans, a G/A polymorphism within exon3 of FATP4 that gives rise to a Gly209Ser substitution is shown to be associated with insulin resistance [38]. The heterozygous carriers of the Ser209 allele show improved metabolic parameters, such as, lower body-mass-index, blood TGs and lipoprotein levels, systolic blood pressure, and insulin concentrations. Hence, FATP4 has been proposed as a key and multifunctional FATP enzyme to be involved in lipid metabolism that leads to human obesity and insulin resistance [38,82,83].

FATP4 in metabolic organs and tissues FATP4 in the gut
Expression pattern of FATP4 in the gut FATP4 is highly expressed in enterocytes or intestinal mucosa reported in many vertebrates including yellow catfish [107,108], chickens [109,110], mice [46], rats [111], pigs [112][113][114], and humans [115]. Interestingly, no significant differences in FATP4 expression are observed between duodenum, jejunum, ileum, proximal colon, and distal colon in human gut [115]. According to the Human Protein Atlas (https://www.proteinatlas.org/), FATP4 expression is relatively lower in colon than other segments of the gut. Nonetheless, FATP4 expression in intestine is higher than in any other organs (Figure 1), and therefore FATP4 in this tissue has been intensively investigated in the past two decades.

FATP4 role on lipid absorption and metabolism in the gut
After the discovery of FATPs during 1994-1998 [28,31], FATP4 was proposed to be a major importer of dietary FAs because of its predominant expression in gut [46,115,116]. Overexpression of FATP4 in cells (HEK293 and primary mouse enterocytes) increases long-chain FA uptake, while FATP4 deficiency inhibits intestinal uptake [46]. At the time, these results suggested that FATP4 could be a principal FA transporter in enterocytes, and that FATP4 inhibitors might be of therapeutic use as anti-obesity drugs. Although mice that are heterozygous for the exon 2 Fatp4 mutation were viable and they showed marked reduction of FA uptake in enterocytes, however they did not show any defects in fat absorption [78]. This finding in 2003 was the first report to undermine the importance of FATP4 in intestinal FA uptake. During 2001-2007, FATP4 was reported to exhibit ACS activities with specificity towards VLCFAs [55,58,62,63]. Thus, FATP4 is thought to be bifunctional as an FA transporter and ACS, and that FATP4-dependent vectorial FA acylation works in concert to mediate FA influx [48,55,[60][61][62][63], which is the same mechanism as FATP1 [49,50]. The lack of effects on intestinal FA absorption in Fatp4-allele deleted heterozygous mice [78] may also suggest some subtle uncharactereized metabolic changes driven by global FATP4 deletion.
During 2006-2014, more detailed molecular studies have confirmed the ER localization of FATP4 in cultured cells [60,117,118]. It is shown that FATP4 works synergistically with the plasma membrane translocase FAT/CD36 in facilitating FA transport via ACS-driven mechanism [117]. Another study has demonstrated that the rescue of Fatp4-null mice with keratinocyte-specific FATP4 overexpression requires an intact ACS domain [119]. As FATP4 overexpression increases ACS activity in hepatoma cells [120], thus the intact ACS domain is necessary for the functions of FATP4 under in vitro and in vivo conditions. While FATP4 exhibits ACS-specific activities with the preference towards VLCFA C24:0 in vitro [60,62], the skin and intestine of Fatp4-null mice also show reduced esterification of C24:0, but not C16:0 or C18:1 [63]. The defects in esterification of VLCFAs to ceramides could be observed in skin Fatp4-null mice, and this event underlies the observed skin abnormalities [76,79,102]. Because Fatp4-allele deleted heterozygous mice did not show any defect in intestinal fat absorption [78], we surmise that Fatp4-dependent ACS activities towards the abundant LCFAs may not lead to an FA uptake, but rather to other metabolic pathways. Taken together, FATP4 indeed acts as an ACS and is not essential for FA absorption in the intestine, and this notion was discussed in 2018 by Cifarelli and Abumrad [121] as well as Schreck and Melzig [122].
In 2009, keratinocyte-specific Fatp4-rescued Fatp4-null mice [119] were used to investigate the effects of FATP4 on lipid absorption after feeding with a Western diet [123]. With an exception for keratinocytes, these mutants would still exhibit Fatp4 deletion in all other cell types, such as, adipocytes, immune cells, muscle cells, hepatocytes, and enterocytes. Results showed no difference in food consumption, growth, and body-weight gain. No intestinal TG absorption and fecal fat losses could be found. Thus, FATP4 is dispensable for intestinal lipid absorption in mice. No difference in hepatic lipid contents and TG secretion could be observed after injection with a lipoprotein lipase inhibitor Tyloxapol. This indicates no FATP4 role on hepatic FA uptake as well. This lack of FATP4 role on lipid absorption is supported by an earlier study in 2006 demonstrating a failure of FATP4-specific inhibitors to attenuate fat absorption in vivo [124]. Despite no difference in intestinal TG absorption kinetics, these mutants fed Western diet showed a significant increase in enterocyte TG and FA contents. This suggests that FATP4 deficiency may shift FA activation towards TG synthesis in intestine. This notion can be supported by other evidence for an increase of neutral lipids including TGs in the skin of IPS patients with FATP4 mutations [75] and Fatp4-null mice [76,79]. Although FATP4 is considered an important protein in lipid absorption research [125], FATP4 deficiency does not per se affect FA uptake, but it may induce a shift of FA metabolism to TGs. Besides intestine, the metabolic changes in other tissues, such as muscle and adipose tissues, may contribute to the lack of effects on blood TG levels in these mutants fed Western diet [123]. Nonetheless, the determination of blood chylomicrons under this condition would have been useful to specifically clarify intestinal fat absorption.
Similar to Fatp4, LCFA ACS-5 (or Acsl5 in the mouse) is also highly expressed in mouse jejunum [126]. While Acsl5 KO mice fed high-fat diet (HFD) displayed a decrease in ACS activity, they did not show any changes in dietary FA absorption and body-weight gain. It is concluded that the residual ACS activity in Acsl5 KO mice is sufficient to maintain normal levels of FA absorption. These results are similar to those from keratinocyte-rescued Fatp4-mutants fed Western diet [123] indicating that only minimal levels of intestinal ACS/FATP enzymes are nescessary for normal FA absorption.
Intestine-specific Fatp4-deficient mice have been generated in our laboratory and our initial report was published in an abstract form [127]. Under normal diet, these Fatp4-deficient mutants showed a decrease in intestinal PLs, sphingomyelin, and ceramides concomitant with an increase in intestinal TGs and CEs. Upon starvation, these mutants showed a moderate increase in blood lipoproteins. Upon feeding with a high-fat high-cholesterol diet, these mutants showed a strong increase in blood lipids and lipoproteins. As shown in Figure 3A, rather than the anticipated inhibition of lipid absorption, intestinal Fatp4 deletion appears to worsen metabolic conditions as seen in elevation of blood lipids and lipoproteins. We propose a shift of FA activation towards TGs in FATP4-deficient intestine, and this event leads to the secretion of chylomycrons by intestine and subsequent secretion of lipoproteins by liver. Similarly to keratinocyte-rescued Fatp4-null mutants [123], an increase in blood lipoproteins including chylomicrons in our intestine-specific Fatp4-deficient mice is not sufficient to elevate hepatic steatosis under dietary stress [127] ( Figure  3A).

The transcriptional factors affecting FATP4 transcription in the gut
A number studies have investigated intestinal expression of FATP4 in vivo, and these studies may provide some clues regarding the regulation of FATP4 in the gut. Intestinal FATP4 expression is not affected by gene deletion of a mucin-generating enzyme [128] or FA uptake enzyme FAT/CD36 [129]. However, FATP4 is down-regulated in the intestine of mice with global deletion of lysophosphatidylcholine acyltransferase3 (LPCAT3) [130] or group VIA calcium-independent phospholipase A2 (iPLA2β) [131]. LPCAT3 catalyzes the acylation of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC). On the opposite, iPLA2β hydrolyzes PLs to lysoPLs at sn-2 position, and it has a greater specificity for hydrolyzing phosphatidylethanolamine (PE) compared with other PLs. This implies that the deletion of either of these genes may lead to changes in intestinal PC and PE that subsequently affect FATP4 transcription and expression. Specifically, it appears that a decrease in intestinal PC/PE ratio by LPCAT3 or iPLA2β deficiency would result in suppressed FATP4 expression. The mechanism for this may involve attenuated activation of an ER transmembrane protein inositol-requiring enzyme1α leading to suppressed transcription of homeostatic genes including FATP4 [131]. Upon inhibiting intestinal cholesterol absorption either by the deletion of Niemann-Pick type C-1 like protein 1 or treatment with a lipid-lowering drug Ezetimibe [132], intestinal FATP4 expression is shown to be suppressed. This suggests a link between FATP4 and cholesterol metabolism in intestine. Fasting for 1-13 days is also shown to down-regulate intestinal FATP4 expression which can be reversed upon refeeding [133]. While short-term 4-day HFD feeding down-regulates intestinal FATP4 expression [134], chronic feeding with fructose [135], HFD [136] or oil [137] however does not alter intestinal FATP4 expression. Thus, intestinal FATP4 appears to be regulated by changes in PC/PE ratio and cholesterol metabolism as well as acute changes of gut nutrients due to starvation and fat exposure time.
We now discuss transcriptional factors that regulate intestinal FATP4 transcription. It has been shown that peroxisome-proliferator-response element is present in the upstream region of FATP gene, and proxisome proliferator-activated receptor (PPAR) agonists such as linoleic acids and PUFAs may transcriptionally regulate FATP4 expression [138]. It has also been shown that the activation of mitogen-activated protein kinase member ERK1/2 induces an inhibition of PPARγ resulting in down-regulation of FATP4 expression in piglet intestine [139]. Thus, ERK1/2 and PPARγ are upstream regulators of FATP4 (Figure 4). It is reported that intestinal Fatp4 expression is not altered by an administration of a PPARα agonist in mice [140]. Thus, PPARγ is a key regulator of intestinal FATP4. Moreover, it is shown that fatty acyl-CoAs are able to suppress PPARγ activation and thus they play an opposing role from FAs [141]. Hence, intestinal FATP4 and its substrate FA and product fatty acyl-CoAs may regulate PPARγ in a feedback-loop manner (Figure 4). Taken together, under dietary stress FATP4 inactivation may increase FAs which are incorporated into intestinal TGs [123] and released as chylomicrons and subsequently lipoproteins [127] (Figure 3A).

FATP4 in fatty liver disease
In mammals, the liver plays an essential role in maintaining metabolic homeostasis of lipids [24]. Compared with FATP2 and FATP5, FATP4 is a minor FATP in the liver [28,31,116]. However, it is shown that FATP4 protein expression is increased in fatty livers in genetic ob/ob and HFD-fed mice [142][143][144]. Since accumulation of hepatic lipids is a common consequence of obesity [145,146], current data support the notion that FA activation by FATP4 may lead to enhanced FA uptake and subsequent metabolism could lead to lipid accumulation in hepatocytes. Similar to fatty liver, FATP4 expression is also increased in adipose tissues of obese women [38,82,83,147] suggesting that TG-FA recycling and mobilization from adipose tissues via TG lipolysis may additionally exacerbate fatty liver development [148][149][150]. As food deprivation in mice is known to induce lipid mobilization from adipose tissues to liver, hepatic steatosis is developed under this condition together with increased hepatic Fatp4 expression [143]. This is in contrast with the reported decrease of intestinal FATP4 expression during fasting [133]. In addition to liver TGs, other deleterious lipid species have been reported to be increased in fatty liver including diacylglycerol and CEs together with a decrease in hepatic PC/PE ratio [145]. Thus, FATP4-mediated FA activation could lead to alterations of various polar lipids and neutral lipids which in turn could play a crucial role for development of MAFLD/NAFLD in vivo.
Mechanistically, FATP4 is localized in the ER and mitochondria of hepatocytes [120,142], which is consistent with the findings in the intestine [115] and other cell types [55,60,61,117,118]. This indicates that FATP4 in the hepatocytes could as well function as an ACS rather than a direct FA transporter. As previously reported in skin fibroblasts [55], fatty acyl-CoAs activated by FATP4 may undergo β-oxidation to ATP and ketone bodies in hepatocytes. By overexpressing FATP4 in human hepatoma cells, a significant increase in ACS activities, FA uptake, cellular lipids, and apoptosis, have been observed [120,142,151]. Collectively, FATP4 functions as an ACS in hepatocytes and its overexpression may contribute to fatty liver disease.
Although forced expression of FATP4 in hepatoma cells leads to an increase in cellular uptake and steatosis [120,142], this model may be applicable to cancer cells and may not be realistic in normal liver in which hepatic FATP4 expression is relatively low [28,31,68,116,138]. The observed high expression of FATP4 in livers of obese mice [142,151] may not per se be a direct cause of fatty liver because FATP4 can be up-regulated by dietary essential FAs known as PPAR agonists [138] (Figure 4). The first clue for no effects of Fatp4 deficiency on hepatic steatosis was already shown in keratinocyte-rescued Fatp4-null mice [123]. These mutants were treated with lipoprotein lipase inhibitor Tyloxapol which results in extremely high blood TGs, and these high levels could mask the much lower levels of metabolically generated TGs by hepatic Fatp4 deficiency. To dissect the functions of hepatic FATP4, mice with hepatocyte-specific Fatp4 deletion were generated in our laboratory.
We published our results in 2019 and demonstrated that hepatocyte-specific FATP4-deficient (Fatp4L -/-) mice under chow diet display no phenotypes regarding body weights and blood lipids [152]. Upon feeding with either a high-sugar or high-fat/high-sugar diet, Fatp4L -/mice showed elevated levels of plasma TGs, non-esterifed free fatty acids (NEFA), and glycerol ( Figure 3B). By combining body and liver weights of female and male mice, Fatp4L -/mice under a high-sugar diet had lower weights, but they were not protected from diet-induced fatty liver. Hence, our data on liver lipids and steatosis are consistent with those from keratinocyte-rescued mutants fed Western diet [123]. Remarkably, an increase in plasma lipids of Fatp4L -/mice fed the diets indicates worsen metabolic hepatic dysfunctions and hyperlipidemia ( Figure 3B).
Since the liver plays an important role in the regulation of blood lipids [24], several FATPs in liver may inherently be involved in this process via FA uptake, FA metabolism, and subsequent secretion of lipids and lipoproteins. Although FATP2 [71,72,120] and FATP5 [80,81,153,154] are recognized as the major FATPs and FA transporters in the liver, hepatic FATP4 appears to be bifunctional, i.e., mediating FA uptake when overexpressed in vitro [120,142] and regulating FA activation and metabolism upon its deletion in vivo [152]. In either condition, FATP4 is involved in the secretion of lipids. The increase in plasma NEFA and glycerol in our mutants fed high-sugar diets suggests an increase in lipolysis activities since the liver contains several intracellular lipases and hepatic lipase at plasma membrane. To investigate this possibility, we generated a stable FATP4-knockdown system in HepG2 cells and published our first results in an abstract form [155]. We observed that lipolysis lipids NEFA, glycerol, and lipoproteins were indeed increased upon FATP4 knockdown, and such increase was further exacerbated by oleate treatment. Thus, TG lipolysis mechanism would be consistent with an increase in blood lipids in Fatp4L -/mice fed with high sugar and fat diets ( Figure 3B). Apparently, TG lipolysis activities are not as efficient in the intestine and skin. This would result in an accumulation in TGs in intestine of keratinocyte-rescued Fatp4-null mice fed Western diet [123] and intestine-specific Fatp4-deficient mice [127], as well as an increase in neutral lipids in the skin of Fatp4-null mice [76,79] and IPS patients [75].

The transcriptional factors affecting FATP4 transcription in the liver
Hepatic FATP4 expression is increased in mouse fatty liver upon feeding with HFD [142,144] or methionine-choline deficient (MCD) [156] diet. The regulation of FATP4 in liver is different from that in intestine whereby lipid metabolism in liver appears to be more robust [5,6]. Regarding FATP4 metabolism, FATP4 deficiency may also lead to changes in FA/fatty acyl-CoA ratio that could alter the activities of PPAR transcription factors in the liver [141,[157][158][159]. Compared with other PPARs, PPARα plays a major role on gene expression in liver [160]. PPARα is shown to up-regulate the transcription of FATP4 and multiple genes which are responsible for lipid metabolism in HepG2 cells and human liver [161,162]. Transcription factors SREBP-1c and NF-κB do not have any effects on FATP4 expression, while they can up-regulate FATP2 and FATP5 expression as well as long-chain FA uptake in the liver [163]. Thus, FATP4 expression may be predominantly regulated by PPARα and PPARγ in the liver and intestine, respectively ( Figure 4). As FAs can modulate FATP4 expression [108,142,143,159], a feedback loop between PPARα and FATP4 may be operative via FAs and fatty acyl-CoAs [141,157,158] (Figure 4). This feedback loop is to some extent auto-regulated to maintain hepatic FA homeostasis by FATP4.
We further discuss the effects of FAs on hepatic FATP4. An initial palmitate or oleate exposure is shown to decrease FATP4 expression [108], and this is thought to be a protective response in preventing FA-overload and toxicity. As FA exposure persists, FATP4 expression in enterocytes is shown to be recovered to basal levels [136]; however, the recovery of FATP4 expression in hepatocytes is found to be greater than basal levels [108,142,143]. We thus propose that FATP4 in normal liver may exhibit a protective function while excessive FATP4 combined with dietary stress is cytotoxic to hepatocytes [142]. Upon FATP4 deletion in hepatocytes under high-sugar and high-fat conditions [152], homeostatic FA metabolism is then shifted towards TGs which are readily hydrolyzed to glycerol and NEFA ( Figure 3B). These blood lipids may in turn lead to toxicity in peripheral tissues leading to metabolic syndromes without any increase in hepatic steatosis, which is known as non-obese MAFLD/NAFLD. The proposed mechanism in Figure 3B indeed supports an association of FATP4 polymorphisms with blood lipids and insulin resistance in humans [38,82,83].

FATP4 role on lipid metabolism in the muscle
Muscle is an important organ in FA metabolism and closely associated with insulin resistance [23,164]. FATP4 is expressed abundantly on the sarcolemma in skeletal muscle [165]. Other reports have shown intracellular FATP4 localization but not at the sarcolemma [118,166]. Similar to FATP4 in the intestine [60] and liver [120,142,152], FATP4 in muscle exhibits high ACS and FA-uptake activities, which are not modulated by insulin [118]. It has been shown that Fatp4, Fat/Cd36, Fatp1, and plasmalemmal FA-binding protein (pm-Fabp) mediate the transport of FAs and also increase β-oxidation in skeletal muscle of rats [167]. None of these four proteins is able to alter the rates of FA esterification into TGs in muscle. Thus, FATP4 catalyzes FAs into fatty acyl-CoAs which are directed to β-oxidation rather than esterification in the muscle.
In humans, skeletal muscle FATP4 protein is increased by ∼30%, while FATP1 protein is reduced by ∼20%, after an 8-week supervised aerobic training [168]. This supports the notion that FATP4 is involved in training-induced FA β-oxidation in skeletal muscle. Compared with FATP1, FATP4 appears to be more specific for channeling of FAs towards β-oxidation. A member of a subfamily of serine/threonine kinase Akt2 is shown to be involved in insulinand contraction-induced FA transport in mouse muscle [169]. Akt2 is also shown to mediate intracellular retention of Fatp4 and pm-Fabp as well as the translocation of Fat/Cd36 and Fatp1 in mouse muscle. Thus, unlike FAT/CD36 and FATP1, FATP4 in the sarcolemma of muscle plays a predominant role in activating FAs for β-oxidation and energy production.

Factors that regulate muscle FATP4 expression
Unlike other metabolically active tissues, obesity and fat intake [170,171] as well as PPARγ activation [172] have little effects on FATP expression in muscle. This is because the muscle expresses very low levels of PPARα and PPARγ [173]. Although PPARα is reported to play a predominant role in skeletal muscle when treated with a PPAR agonist [160], there has been no investigations on its relationship with FATP4 expression in skeletal muscle. The transcriptional regulation of FATP4 in muscle cells is still elusive. However, muscle FATP4 expression is increased upon contraction by excercise [66,174] or electric-pulse stimulation [175] in mice. Similarly, the sensitivity of muscle FATP4 is enhanced by insulin [66,118]. FATP4 may thus facilitate insulin sensitivity for muscle FA β-oxidation. As muscle mass is regulated by myostatin, the deletion of this gene increases muscle FATP4 expression which leads to muscle hypertrophy together with reduction of body fat accumulation [176]. The deletion of a zinc transporter znt7 in mice also increases muscle FATP4 expression, and this results in lean phenotypes thus highlighting an indirect role of zinc on muscle FA β-oxidation [177]. During plamitate stress in C2C12 myotubes, vascular endothelial growth factor B treatment is shown to reduce lipid droplet accumulation concomitant with upregulation of FATP4 [178]. Hence, ample evidence supports the role of FATP4 in FA β-oxidation muscle. In contrast with FATP4, FATP1 in muscle appears to mediate lipid deposition and subsequent insulin resistance [179]. Thus, FATP4 may serve as a potential target for improving insulin resistance in muscle.

FATP4 role on lipid metabolism in adipose tissue
Adipose tissue is the main lipid storage of the body, and adipocytes control flux of FAs to peripheral tissues by storing and hydrolyzing TGs under hormonal controls [180,181]. It has been shown that FATP1 and FATP4 expression and FA-uptake activities are increased following insulin-induced differentiation of mouse 3T3-L1 fibroblasts to adipocytes in part by promoting membrane trafficking of intracellular FATP1 and FATP4 to the plasma membrane [47]. Moreover, TNF-α, which induces insulin resistance via tyrosine hypophosphorylation of insulin receptor [182], has been shown to inhibit adipocyte expression of FATP1 and FATP4 [183] and attenuate insulin-induced long-chain FA uptake in adipocytes [184]. Hence, FATP1 and FATP4 may be the major effectors in the regulation of FA flux into and out of adipocytes exerted by insulin [47,185]. These two enzymes may thus play an important role in energy homeostasis and metabolic disorders in adipose tissue. However, an opposite finding has been reported for endothelial cells. An increase in FATP4 expression was observed during TNF-α-stimulated endothelial autophagy and NF-κB signaling as well as palmitate-induced transcytosis across microvascular endothelial cells [186]. Thus, FATP4 is differentially regulated by TNF-α in adipocytes versus in endothelial cells.
In acquired obesity in humans, FATP4 expression is up-regulated in adipose tissues and closely correlated with increased body-mass-index, body-fat mass, and insulin resistance [82,83,147,187]. Genetic polymorphisms of FATP4 are associated with metabolic parameters in humans [38] as well as body-fat mass in pigs [188] and chickens [110]. FATP4 and FAT/CD36 expression as well as TG synthesis in adipose tissue of African-American women are higher than those of Caucasian women [147,187]. These findings suggest a significant role of FATP4 in adipose fat accumulation and metabolism in vertebrates.
An earlier study has shown that FATP4 knockdown in adipocytes has no effects on preadipocyte differentiation and FA influx but markedly increases basal lipolysis resulting in reduced cellular levels of TGs [67]. In the context of TG lipolysis, we have consistently shown that the levels of plasma glycerol and NEFA are further elevated in adipose-specific Fatp4 knockout (Fatp4A -/-) mice fed with methionine-and choline-deficient (MCD) diet [189] ( Figure 3C). It is known that methionine-and choline-deficiency in cultured adipocytes and in mice fed with MCD diet triggers an induction of TG lipolysis detectable in culture medium and blood, respectively [149,150]. Consistently under ex vivo conditions, an exposure of mouse adipose tissues to MCD medium increased the secretion of glycerol and NEFA, and this secretion was further increased by adipose FATP4 deficiency [189]. Fatp4A -/mice under chow diet already showed an increase in blood lipoproteins and leptin but a decrease in β-oxidation products, ketone bodies. These mutant mice when fed with MCD diet showed a further elevation of plasma leptin with suppression of plasma ketone bodies. Thus, ketone bodies may be regulated by leptin by adipocyte FATP4 deficiency in vivo ( Figure  3C). The mechanism for an increase in leptin by the deficiency is not known and speculatively may involve an increase in malonyl-CoA [189]. Despite elevated blood glycerol and NEFA, an increase of leptin seen in MCD-fed mutants may in turn attenuate hepatic steatosis ( Figure 3C). Despite steatosis protection, liver fibrosis is not protected. Thus, adipose FATP4 deficiency during MCD-induced non-obese MAFLD leads to metabolic disorders with elevated blood lipids in a similar manner as hepatocyte Fatp4 deficiency under high-sugar/high-fat conditions ( Figure 3B,C).
In 2011, our first study on Fatp4A -/mice was carried out in obese NAFLD model by HFD feeding for 12 weeks [190]. Upon intragastric loading of 9,10-3 H labelled palmitate or 1-14 C labelled lignocerate, there was no difference in flux activity in white adipose tissues between control and Fatp4A -/mice. This indicates that adipose FATP4 does not play any role in FA uptake in vivo. Upon HFD feeding, Fatp4A -/mice showed a significant increase in body weights, adipocyte expansion, and the development of fatty liver. Lipidomics analysis showed a decrease in subcutaneous fat PLs and SPLs concomitant with an increase in TGs. Thus, for adipocytes the shift of FA from PLs and SPLs towards TGs was already observed in 2011 ( Figure 3D). Taken together, adipose Fatp4 deficiency elicits an opposing effect in suppressing and exacerbating hepatic steatosis in the non-obese MCD ( Figure 3C) and obese HFD ( Figure 3D) model, respectively.
Since aP2-Cre (or Fabp4-Cre) mouse line was used to generate Fatp4A -/mice, it has been a concern whether these mutants could also exhibit FATP4 deletion in macrophages because aP2/Fabp4 is also highly expressed in macrophages [191]. Therefore, we generated macrophage-specific Fatp4-deficient mice (Fatp4M -/-) by cross-breeding LysM-Cre with Fatp4flox mouse line [192]. Bone marrow-derived macrophages (BMDMs) from control and Fatp4M -/mice were utilized to investigate ER stress response in vitro [192]. Under basal levels, Fatp4M -/-BMDMs showed a decrease in cellular sphingomyelin and 24:1 ceramides indicating FATP4 role in the syntheses of SPLs and VLCFA-containing ceramides. These results are consistent with a previous report in keratinocytes [76,102]. ER stress in control BMDMs suppressed the levels of SPLs which were further suppressed by FATP4 deficiency. During ER stress, the deficiency also caused a further increase in cellular TGs. Thus, these results are again consistent with FA metabolic shift towards TGs as seen in FATP4-deficient enterocytes, hepatocytes, and adipocytes under dietary stress ( Figure 3). Collectively, our data have demonstrated a fine balance between polar lipids (e.g., PLs and SPLs) and neutral lipids (e.g., TGs and CEs) regulated by FATP4 in intestine [123,127], liver [142,152,155], adipocytes [189,190], and macrophages [192]. Via TG metabolism, FATP4 deficiency causes worsen metabolic outcomes with elevated blood lipids and exaggerated fatty liver disease in diet-induced obese MAFLD/NAFLD (Figure 3).
Mechanistically, FATP4 localized in the ER of adipocytes is shown mediates FA uptake indirectly [60,61]. FATP4 is present only in detergent-soluble membranes but not in detergent-resistant membranes (lipid-raft domains) of adipocytes [193,194]. As FAT/CD36 localized at the plasma membrane is the main adipocyte FA transporter, FATP4 is not co-localized with FAT/CD36 nor directly interacts with lipid-raft associated FAT/CD36. This highlights the function of FATP4 in adipocytes as an intracellular ACS in mediating FA uptake by metabolic trapping and balancing polar and neutral lipids during dietary stress in non-obese ( Figure 3C) and obese ( Figure 3D) MAFLD/NAFLD models.

Transcriptional factors affecting FATP4 transcription in adipose tissue
Upon depletion of a histone demethylase, FATP4 expression in adipose tissues is decreased resulting in impaired adipogenesis [195]. These data suggest epigenetic mechanism for regulation of adipose FATP4. As biotin attenuates many parameters of metabolic syndrome disease, biotin supplementation is shown to up-regulate adipose FATP4 expression [196]. This up-regulation may result in suppression of adipose TG lipolysis [67] hence improving hyperlipidemia and metabolic syndrome. A flavonoid derivative Aaicalein has been developed as an activator of peroxisomal β-oxidation [197]; however, this compound does not have any effects on FATP4 expression in skin fibroblasts from X-ALD patients [198]. These data also support the notion that FATP4 is not the key enzyme but acts as a co-factor for VLCFA β-oxidation in X-ALD [90].
Here, we discuss the transcriptional factors that regulate adipocyte FATP4 transcription. It has been shown that FATP genes are regulated by PPARα and PPARγ in 3T3-L1 adipocytes [138]. In white adipose tissues of obese mice, PPARγ activators such as pioglitazone and troglitazone, have been shown to increase the expression of FATP genes more efficiently than PPARα activator clofibrate [199]. PPARγ ligand also induces FATP expression in 3T3-L1 adipocytes quicker than PPARα activator [173]. A correlation between PPARγ and FATP4/CD36 expression in human adipose tissues has also been reported [147]. Thus, PPARγ is the major regulator of FATP4 expression in human and mouse adipocytes (Figure 4). This is consistent with the observations in intestine in which PPARγ and FATP4 are highly expressed [112]. Thus, FATP4 is regulated by PPARs in hepatocytes [162] and adipocytes [173,199,200]. While PPARγ regulates FATP genes [138], knockdown of FATP4 in 3T3-L1 adipocytes on the other hand leads to an increase in PPARγ expression [67]. Knockdown of ACSL1 in human hepatocytes is also shown to increase PPARγ expression [201]. Similar to the events in enterocytes and hepatocytes, FATP4-mediated changes in FAs and fatty acyl-CoAs [141,157,158] could regulate PPARγ in a feedback-loop manner in adipocytes (Figure 4).

Conclusions
FATP family members are a class of enzymes involved in FA activation and metabolism and they have significant contribution on the maintenance of lipid homeostasis. Among these members, FATP1 and FATP4 are the only two evolutionary conserved genes from invertebrates to vertebrates. FATP4 is the only member whose mutations or deletion can cause death in humans and animals. FATP4 was originally considered as an FA transporter, it has been now well accepted as an ACS. While the skin is the organ most affected by FATP4 mutations or deficiency, accumulating evidence has indicated a significant role of FATP4 in other metabolically active tissues, such as, intestine, liver, muscle, and adipose tissues. The evidence against FATP4 as an FA transporter has been published by several investigators including us. The up-regulation of FATP4 expression in adipose tissue with acquired obesity and in the liver with MAFLD/NAFLD indicates an involvment of FATP4 in these metabolic diseases. This is supported by association of FATP4 polymorphisms with body-mass index, body-fat thickness, and blood TGs. Our research work using tissue-specific Fatp4-deficient mice has demonstrated a significant role of FATP4 in cellular metabolism of polar and neutral lipids, and that its deficiency leads to a metabolic shift towards TGs, lipoproteins, and lipolysis products ( Figure 3). Our research results highlight FATP4 as a model ACS that regulates lipid metabolism not only in skin but also metabolically active tissues. The worsen metabolic outcomes observed in tissue-specific Fatp4-deficient mice may be applicable to patients with FATP4 mutations who comsume high-sugar and/or high-fat diets.


s2fieldsofstudy: Biology Medicine

text:
Association between CILP and IL-1α polymorphisms and phenotype-dependent intervertebral disc degeneration susceptibility: A meta-analysis

Background: The relationship between CILP (1184T>C) and IL-1α(+889C/T) polymorphisms and intervertebral disc degeneration (IDD) have been explored in several studies but the results were conflicting. The aim of the study was to evaluate and synthesize the currently available data on the association between CILP (1184T>C) and IL-1α(+889C/T) polymorphisms and susceptibility of phenotype-dependent radiologic IDD (RIDD) and symptomatic intervertebral disk herniation (SIDH). Methods: A computerized literature search was in PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure database, and Web of Science. The pooled results were presented as odds ratios (ORs) with 95% confidence intervals (CIs). Moreover, the false-positive report probability (FPRP) test and trial sequential analysis (TSA) were applied to estimate the significant results. Results: Our evidence demonstrated that IL-1α(+889C/T) was significant associated with RIDD (allele model: OR = 1.34, 95%CI 1.03–1.74, p = 0.029) and SIDH (allele model: OR = 1.28, 95% CI 1.03–1.60, p = 0.028). However, the results were not noteworthy under the FPRP test and TSA analysis. Additionally, CILP (1184T>C) polymorphism was significantly associated with RIDD with adequate evidence (allele model: OR = 1.27, 95% CI 1.09–1.48, p = 0.002) instead of SIDH. Conclusion: The current meta-analysis illustrated firm evidence that CILP (1184T>C) polymorphism was significantly associated with the susceptibility of RIDD. However, the significant associations between IL-1α(+889C/T) and RIDD and SIDH were less credible. Thus, more multi-center studies with diverse populations were required to verify the results.

Introduction
Low back pain (LBP), one of the most common muscleskeletal diseases, contributes to lower life quality and even disability. Over 80% of elderly people are afflicted with LBP to some extent during their lifetime (Andersson, 1999). Intervertebral disc degeneration (IDD) is regarded as a major cause of LBP which has an extremely complex pathogenesis (Chou et al., 2011). Multiple risk factors such as environmental, diet, and genetic factors were reported to accelerate the pathogenesis process of IDD (Battié and Videman, 2006).
As a chondroid structure, the nucleus pulposus (NP) contains a luxuriant extracellular matrix (ECM) such as proteoglycans and collagen. Accumulating evidence has highlighted the important role of ECM homeostasis play in the development of IDD. The imbalance of ECM homeostasis may cause diverse changes in disc including height reduction, dehydration, endplate alteration, etc. (Roberts et al., 2006). Overexpression of proinflammatory and catabolic phenotypes is the major reason for the imbalance of ECM homeostasis in the NP (Sivan et al., 2014;Molinos et al., 2015). Overexpression of IL-1 in the degenerative disk tissues inhibited extracellular matrix synthesis and improved extracellular matrix degradation (Phillips et al., 2013;Li et al., 2017). Hypersensitivity to IL-1α in disc cells is a significant motivator for degeneration (Maeda and Kokubun, 2000). Cartilage intermediate layer protein (CILP) is sufficiently distributed in intervertebral discs and has a vital function in regulating the metabolism of ECM. CILP inhibits the production of cartilage matrix protein via the TGF-β1signaling pathway (Seki et al., 2005).
Given the vital function of the IL-1α and CILP in ECM metabolism, numerous candidate polymorphism loci correlated to IDD predisposition were identified. Various studies indicated that CILP (1184T>C) and IL-1α(+889C/T) polymorphisms could be significantly correlated with the susceptibility of IDD (Solovieva et al., 2004;Seki et al., 2005). However, recent studies failed to detect the association and even obtain the opposite conclusion (Virtanen et al., 2007;Kelempisioti et al., 2011;Abdollahzade et al., 2018;. More importantly, the results of previous systematic reviews are unreliable due to overlooking the phenotypes of IDD (Wang W. et al., 2016;Wang Z. et al., 2016;Rajasekaran et al., 2016). Under the circumstances, we aim to provide more exhaustive evidence and rigorous criteria to explore the correlation of IL-1α and CILP polymorphisms with radiologic IDD (RIDD) as well as symptomatic intervertebral disk herniation (SIDH).

Search strategy for literature
We performed a literature retrieval of PubMed, China National Knowledge Infrastructure database, Web of Science, Cochrane Library database, and Embase by using the following terms ("LDD" or "IDD" or "lumbar disease" or "lumbar degeneration" or "Intervertebral disc degeneration" or "herniation") AND ("polymorphism" or "allele" or "variant") AND ("IL-1α" or "CILP" or "IL-1A"). The bibliography of associated studies was also examined for additional correlative studies.

Selection criteria
Studies included in the meta-analysis should be based on the following criteria: 1) case-control study; 2) the consequence was the investigation of the correlation between CILP (1184T>C) IL-1α(+889C/T) or rs1800587 or rs2073711and IDD or SIDH; 3) genotype frequencies were available to calculate an odds ratio (OR) with 95% confidence intervals (CIs); 4) grade criteria or phenotypes of IDD were described in detail.
The exclusion standards were listed as follows: 1) other types of study design; 2) genotype frequencies were not present in the articles; 3) studies contained overlapping data from previous research; 4) publication language was not English.

Methodological quality assessment
The quality evaluation of each study was performed independently by two investigators (JCL and XQS) following the Newcastle-Ottawa Scale (NOS). Dissension between reviewers would be discussed to reach a consensus. Based on the NOS standard, only studies with calculating scores >5 were deemed of high methodological quality.

Data extraction
Two reviewers independently extracted the following information on basis of a standard form: 1) Name of the First author 2) Publication information 3) Country 4) Ethnicity 5) Genotype frequencies 6) Numbers of cases and controls 7) Phenotypes of IDD.
Frontiers in Genetics frontiersin.org

FIGURE 1
Flow diagram of the study identification and selection process.

Statistical analysis
Stata 14.0 was applied to conduct the statistical analysis. The correlation strength was assessed by ORs and corresponding 95% Cl in five genetic models, including allele, domain, recessive, heterozygote, and homozygote models. THE Cochrane Q test and the I2 statistic test were performed in each model to assess the heterogeneity between studies, and significant heterogeneity was considered when I 2 >50% or p < 0.05 (Higgins and Thompson, 2002). When significant heterogeneity existed, the pooled ORs would be estimated in the random-effects model; otherwise, the fixed-effects model (Mantel-Haenszel) was selected. Hardy-Weinberg equilibrium (HWE) was utilized to explore the genotype distribution of the population included in this research. Significant disequilibrium was defined by p < 0.05 in the chi-square test. Publication bias was quantitatively assessed through the Begg's and Egger's test and p < 0.05 represented a significant public bias. To evaluate the robustness of overall findings, sensitivity analysis was performed. Subgroup analysis was performed in RIDD to evaluate the genetic effect of IL-1α(+889C/T) and CILP (1184T>C)in different phenotypes of RIDD meanwhile stratified analysis was conducted in SIDH to test the genetic effect of CILP (1184T>C) in different ethnicity. False-positive report probability (FPRP) tests were performed to correct all significant pooled ORs. According to the calculating Excel spreadsheet (Wacholder et al., 2004), the values of FPRP were estimated at the prior probability of 0.01 to detect ORs of 1.5. FPRP >0.2 was thought to be a false significant result.

Trail sequential analysis
In order to control type I and type II errors and predict the required information size (RIS) in the research, the trial sequential analysis (TSA) was carried out in the TSA software (version 0.9; www.ctu.dk/tsa) with a 5% type I error and 20% type II error (Roshanov et al., 2017). The trial sequential monitoring boundaries were based on the O'Brien-Fleming-spending function. If the cumulative Z-curve exceeds the RIS or TSA line, it suggested the significant association is credible. Otherwise, further studies are required to confirm the result (Wetterslev et al., 2017). If the cumulative Z-curve passes the futility boundary, the association is a true negative result.

Study characteristics
The detailed literature search process was present in Figure 1. After duplicate removal and records screening, 25 articles were subjected to full-text screening. Conforming to the selection criteria, 13 studies were excluded with 12 literature remaining. Among them, six recruited studies evaluated the relationship between IL-1α rs1800587 and the risk of RIDD and SIDH in Caucasian, Mexica, and Chinese populations. Six studies consisting of Japanese, Chinese, Finland, and Indian populations were included in the analysis for CILP rs2073711. The characteristics and genotype frequencies of each study were shown in Table 1 meanwhile quality assessment was listed in Supplementary Table S1.

CILP (1184T>C) polymorphism and SIDH susceptibility
A total of 898 cases and 1111 controls (Seki et al., 2005;Virtanen et al., 2007; were recruited and the pooled results demonstrated no significant relationship in any models (Table 3) (Figure 4). All models suggested high heterogeneity. Subgroup analysis revealed a significant association between CILP (1184T>C) polymorphism and SIDH risk in the Asian population. Frontiers in Genetics frontiersin.org

Publication bias and sensitivity analysis
A ruling one out in turn strategy was adopted to estimate the effect of exclusion for each study on pooling ORs. The overall estimates did not alter under any genetic model when each individual study was omitted in turn, demonstrating the robustness of the findings (Figure 6). Begg's and Egger's tests indicated the results were free from

Significance test and TSA results
We performed the FPRP test to investigate the false-positive results.
The relationship between IL-1α(+889C/T) polymorphism and IDD and SIDH was not noteworthy (FPRP >0.2) in any model while the significant association between CILP (1184T>C) polymorphism and RIDD was considered a true positive result. The cumulative z-curve did not pass the TSA and RIS line for the association between IL-1α(+889C/T) polymorphism and IDD, indicating more studies are required (Figure 7). For IL-1α(+889C/T) polymorphism and SIDH, the cumulative z-curve crossed the futility line in the allele model and did not exceed the TSA and RIS boundary in the recessive and heterozygous model (Figure 8), which suggested the association turned out to be invalid in the allele model and more studies are required for the recessive and heterozygous model. Regarding CILP (1184T>C) polymorphism and RIDD, the cumulative z-curve reached the TSA line in the allele and recessive model (Figure 9). The evidence is adequate to confirm this significant association.

Discussion
AS a multifactorial disease, IDD is thought to be mainly caused by overloading and senescence (Rannou et al., 2004;Zhang et al., 2009). In a broad sense, IDD contains multiple potential phenotypes such as SIDH, asymptomatic disc herniation, disc bulge, disc height or signal change, and Modic change. SIDH characterized by sciatica represents a more serious phenotype of IDD because over 70% of individuals with disc herniations are asymptomatic (Boden et al., 1990). Recently, mounting evidence highlights the function of genetic factors in the initiation and progression of Frontiers in Genetics frontiersin.org IDD. TT genotype for IL-1α rs1800587 induced the overexpression of IL-1 (Dominici et al., 2002). The relationship between IL-1α rs1800587 polymorphism and IDD susceptibility has been investigated in numerous studies, but the results were inconsistent. Karppinen et al. and Chen et al. found that IL-1α polymorphism (rs1800587) was associated with the susceptibility of IDD in Finland and Chinese populations (Karppinen et al., 2009;Chen et al., 2018). However, the findings were not detected in Caucasian and Iranian populations (Paz Aparicio et al., 2011;Abdollahzade et al., 2018). CILP (1184T>C) polymorphism resulted in inhibition of the extracellular matrix proteins synthesis (Seki et al., 2005;Min et al., 2010). However, updated studies failed to detect the statistical significance in the Chinese and Finland populations (Virtanen et al., 2007). In addition, studies revealed the C allele as a protective factor in Finnish and Indian populations, which contrasts with studies in the Japanese population (Kelempisioti et al., 2011;Harshitha et al., 2018). Thus, updated research with new evidence was performed to obtain a constructive conclusion. In our meta-analysis, significant associations were detected between IL-1α rs1800587 polymorphism and IDD and SIDH but more evidence is required to confirm the conclusion. The subgroup analysis confirmed that IL-1α rs1800587 polymorphism was correlated with the risk of both RIDD (Solovieva grade) and SIDH but not RIDD (Modic change). Thus, we assumed that the change of expression due to rs1800587 polymorphism might mainly participate in the degeneration of NP instead of the endplate. Moreover, the homozygous model had the highest risk score. Compared with RIDD, a stronger association was detected between IL-1α(+889C/T) and SIDH. The results highlighted the importance of IL-1α polymorphism in the occurrence and progression of IDD.
Moreover, the pooled ORs and 95% CIs and TSA result demonstrated that CILP (1184T>C) was a credible susceptibility variant for RIDD (Pfirrmann grade) instead of SIDH, indicating the genetic effect involved in the initial stage of NP degeneration. Consistent with the results of IL-1α(+889C/T), the highest risk score was detected in the homozygous model. Furthermore, CILP rs2073711 is associated with SIDH in an ethnicity-dependent manner, which was mainly due to the difference in allele frequencies Frontiers in Genetics frontiersin.org for ethnicity. The C allele was found to be a protective factor for Caucasians but a risk factor for Asians. Additionally, the C allele is also a protective factor for knee arthritis in the European population (Valdes et al., 2004), which is consistent with this conclusion. To our best knowledge, this research is the first to comprehensively assess the genetic effect of IL-1α(+889C/T) and CILP (1184T>C) in different phenotypes of IDD. The significant associations were evaluated using the FPRP test and TSA. All eligible data was extracted to provide more convincing evidence. Our findings provided a novel perspective on the relationship between genetic polymorphisms of IL-1α and CILP and IDD phenotypedependent susceptibility and emphasized its role in IDD early screening and SIDH prevention. Population with high-risk genotypes should avoid longtime overloading and other risk factors leading to the development of IDD.
However, limitations should be claimed. First, despite a larger sample size in this study compared with previous studies, samples with other phenotypes of IDD are required to estimate the false-positive result. Second, this study did not estimate multiple confounding factors such as gender, occupation, and environmental factors, which might affect the effect estimates. Third, candidate gene studies have limitations in dealing with complex disorders and the results are often unrepeatable (Arango, 2017). A large-scale meta-analysis is a promising approach to solving the defect. Additionally, the potential value of polygenic risk score analysis for complex diseases has been highlighted (Arango, 2017). Even so, it is crucial to conduct individual studies because individual datasets are the basis of large-scale research and certain hypotheses may be better addressed in smaller studies, at least initially (Thompson et al., 2017).

Conclusion
The current meta-analysis illustrated adequate evidence that CILP (1184T>C) polymorphism was significantly Frontiers in Genetics frontiersin.org associated with the susceptibility of RIDD. However, the significant associations between IL-1α(+889C/T) and RIDD and SIDH were not noteworthy. Further research with larger sample size and various populations is required to verify the outcome.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions
JL participated in designing the experiments, data extraction, data analysis, and drafting the manuscript. YC and XS participated in the data extraction. HW designed and revised the article. All authors approved the final version.