αvß3-targeted sEVs for efficient intracellular delivery of proteins using MFG-E8.

BACKGROUND: Small extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules. RESULTS: In this study, we proposed a membrane-associated protein, milk fat globule-epidermal growth factor 8 protein (MFG-E8), to produce αvß3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)-like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvß3 and αvß5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs' target to integrin αvß3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvß3 positive cells and αvß3 negative cells. Analysis showed higher target protein could transfect into αvß3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvß3 positive cells. CONCLUSION: This study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvß3 positive cells.

Milk fat globule epidermal growth factor 8 (MFG-E8) on monocytes is a novel biomarker of disease activity in systemic lupus erythematosus.

BACKGROUND: Milk fat globule epidermal growth factor (MFG-E8) is related secreted protein which links phosphatidylserine on apoptotic cells and integrin αvß3/5 on phagocytes. To clarify the clinical significance of MFG-E8 in SLE, we analyzed the correlation between expression level of MFG-E8 in circulating phagocytic leukocytes and clinical parameters of patients. METHODS: The study was conducted under a multi-center, prospective cohort design. Patients with one or both BILAG A or B, or SLEDAI- 2 K ≥ 4 with clinical symptoms were defined as the active SLE group. Expression of MFG-E8 on monocytes and concentration in serum were measured by FACS and ELISA, respectively. RESULTS: 96 subjects were enrolled. The absolute number and proportion of MFG-E8-positive monocytes to total monocytes were significantly higher in the active SLE group (p < 0.01). Importantly, the proportion was also significantly correlated with SLEDAI-2K, clinical SLEDAI, as well as serum levels of anti-ds-DNA antibody and complement and C1q. In addition, the proportion of MFG-E8-positive monocytes to total monocytes was significantly decreased from baseline in active SLE patients after 6 months' treatment and increased concordantly with disease activity in 6 refractory cases. Further, in receiver operating characteristic curve analysis for discrimination between active and inactive SLE, the AUC of the proportion of MFG-E8 was 0.854, which was equivalent to classical activity markers such as anti-ds DNA antibody (0.776), complement (0.897) and C1q (0.815). CONCLUSIONS: The proportion of MFG-E8-positive monocytes to total monocytes in peripheral blood was positively associated with disease activity in SLE and may be a novel biomarker of disease activity.

An injectable recombinant human milk fat globule-epidermal growth factor 8-loaded copolymer system for spinal cord injury reduces inflammation through NF-κB and neuronal cell death.

Spinal cord injury (SCI) is a common disease and a major cause of paralysis, carrying much burden around the world. Despite the progress made with growth factors therapy, the response rate of acute SCI treatment still remains unsatisfactory, due largely to complex and severe inflammatory reactions. Herein, we prepare a MFG-E8-loaded copolymer system-based anti-inflammation therapy for SCI treatment. It is shown that the MFG-E8-loaded copolymer system can decrease pro-inflammatory cytokine expression and neuron death. In a rat model of crush-caused SCI, the copolymer system shows significant therapeutic efficacy by ameliorating inflammation, decreasing fibrotic scar, promoting myelin regeneration and suppressing overall SCI severity.

Recombinant Human Milk Fat Globule-Epidermal Growth Factor 8 Attenuates Microthrombosis after Subarachnoid Hemorrhage in Rats.

BACKGROUND: Microthrombosis after subarachnoid hemorrhage has an adverse effect on prognosis. Milk fat globule-epidermal growth factor 8 promotes phagocytosis of phagocytic cells and may reduce microthrombosis. This study investigated the effects of recombinant human milk fat globule-epidermal growth factor 8 on microthrombosis and neurological function after subarachnoid hemorrhage. METHODS: Rats subarachnoid hemorrhage model was induced by intravascular puncture method. Western blot was performed to measure the expression of endogenous milk fat globule-epidermal growth factor 8 after subarachnoid hemorrhage. Microthrombosis was quantified by microthrombi count using immunohistochemistry and immunofluorescence. The neuroprotective effect of recombinant human milk fat globule-epidermal growth factor 8 administration was evaluated by modified Garcia score, beam balance, Rotarod test, and Morris water maze. RESULTS: Endogenous milk fat globule-epidermal growth factor 8 protein level increased after subarachnoid hemorrhage. Microthrombosis was significantly increased in subarachnoid hemorrhage rats brain, while recombinant human milk fat globule-epidermal growth factor 8 dramatically reduced microthrombosis as well as improve short- and long- term neurobehavior after subarachnoid hemorrhage. CONCLUSIONS: Recombinant human milk fat globule-epidermal growth factor 8 reduces microthrombosis and improves neurological function after subarachnoid hemorrhage, which may be an effective strategy for treating subarachnoid hemorrhage.

MFG-E8/integrin ß3 signaling contributes to airway inflammation response and airway remodeling in an ovalbumin-induced murine model of asthma.

Asthma is the most common chronic childhood disease worldwide, characterized by airway remodeling and chronic inflammation, orchestrated primarily by Th2 cytokines. The aim of the current study was to explore the influences of milk fat globule epidermal growth factor 8 (MFG-E8)/integrin ß3 signaling involved in airway inflammation and remodeling in asthma. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. The levels of MFG-E8 expression were declined markedly in the OVA-induced allergy murine model. In addition, administration of MFG-E8 strongly reduced the accumulation of T-helper type 2 (Th2)-associated cytokines (such as interleukin-4, -5, and -13) as well as chemokine CCL11 (eotaxin) in bronchoalveolar lavage fluid and tissues in the OVA-sensitized mice. Moreover, MFG-E8 remarkably repressed the total immunoglobulin E and OVA-specific immunoglobulin E in serum in OVA-challenged mice. Meanwhile, treatment with recombinant murine MFG-E8 noticeably prevented inflammatory cell infiltration into the airways, as showed by a marked decrease in the numbers of total immune cells, eosinophils, neutrophils, macrophages, and lymphocytes in the bronchoalveolar lavage fluid in response to OVA challenge. Importantly, MFG-E8 apparently alleviated OVA-driven airway remodeling, which were evidenced by declined secretion of important mediators of airway remodeling, including transforming growth factor-ß1, matrix metalloproteinase 9, ADAM8, and vascular endothelial growth factor, and reduced airway collagen deposition and inhibited goblet cell hyperplasia in OVA-induced asthma in mice. Mechanistically, integrin 3 contributes to the protective effect of MFG-E8 in inhibiting airway inflammation and remodeling in OVA-driven features of allergic asthma. Overall, MFG-E8, as a candidate molecule to evaluate airway inflammation and remodeling, could be a potential target for the management and prevention of asthma exacerbations, suggesting that MFG-E8/integrin ß3 signaling may serve as a promising therapeutic agent for childhood asthma.

Elevated serum milk fat globule-epidermal growth factor 8 levels in type 2 diabetic patients are suppressed by overweight or obese status.

Inflammation is the most important link between obesity and type 2 diabetes (T2D). Although milk fat globule-epidermal growth factor 8 (MFG-E8) is a key mediator in anti-inflammatory responses, its role in obesity and diabetes is not yet completely understood. We aimed to measure MFG-E8 serum levels and to explore the role of MFG-E8 in obesity and T2D. Fasting serum MFG-E8 levels were quantified by enzyme-linked immunosorbent assay for 168 individuals, whose oral glucose tolerance test was conducted, and levels of inflammatory factors, including tumor necrosis factor-α (TNF-α) and C-reactive protein, were measured. The participants were subdivided into 66 newly diagnosed T2D individuals, 44 impaired glucose tolerance (IGT) subjects and 58 healthy controls. Their characteristics were further classified as lean or nonlean for investigation. MFG-E8 levels were significantly higher in T2D subjects than in healthy controls (P = 0.028). Decreased levels of MFG-E8 were found in overweight or obese individuals, compared to those in lean subjects, in both the T2D and IGT groups (P < 0.001). Interestingly, MFG-E8 levels showed a negative correlation with body mass index (BMI) and TNF-α levels in the total population and the T2D subgroup. Further, BMI and TNF-α concentrations were found to be independent predictors of MFG-E8 levels in all subjects. MFG-E8 levels are elevated in T2D but suppressed by increased adipose tissues, thereby allowing inflammatory factors to rise to high levels. MFG-E8 may serve as a potential biomarker for obesity and T2D in the clinical setting. © 2017 IUBMB Life, 69(2):63-71, 2017.

MFG-E8 overexpression promotes colorectal cancer progression via AKT/MMPs signalling.

Several studies have revealed that MFG-E8 (milk fat globule-epidermal growth factor 8) is related to tumour development and progression. However, the relationship between MFG-E8 expression and metastasis in colorectal cancer patients and the role of MFG-E8 in colorectal cancer invasion and progression remain unknown. In this study, we performed immunohistochemistry and quantitative real-time polymerase chain reaction to assess MFG-E8 expression in colorectal cancer and adjacent non-cancerous tissues. Colorectal cancer RNAseq data from The Cancer Genome Atlas project were downloaded and MFG-E8 expression was analysed. Gene set enrichment analysis was performed for gene ontology and pathway analysis associated with MFG-E8 expression. For in vitro studies, we used lentivirus-mediated MFG-E8 RNA interference and commercialized recombinant human MFG-E8 to investigate its role in colorectal cancer cell growth, migration and invasion. It seems that MFG-E8 was overexpressed in advanced colorectal cancer tissues compared with early-stage colorectal cancer tissues and adjacent non-cancerous tissues. Correlation analysis revealed that MFG-E8 expression was significantly related to plasma membrane invasion, lymph node metastasis, distant metastasis and tumour-node-metastasis stage. Survival analysis revealed that high MFG-E8 expression predicted a poorer prognosis than low MFG-E8 expression group both in our colorectal cancer cohort and The Cancer Genome Atlas colorectal cancer cohort. In vitro study suggested that MFG-E8 knockdown can suppress the growth of colorectal cancer cells without affecting the expression of the proliferation-related gene Ki67. MFG-E8 knockdown also suppressed colorectal cancer cell migration and invasion, a change accompanied by MMP-2 and MMP-9 downregulation. Moreover, MFG-E8 knockdown induced a shift from mesenchymal makers to epithelial makers, while pretreatment with rhMFG-E8 had the opposite effect. The effect of MFG-E8 on colorectal cancer cell migration, invasion and epithelial-to-mesenchymal was partially dependent on the PI3K/AKT signalling pathway. These findings provide a better understanding of the molecular mechanism underlying colorectal cancer progression and suggest a predictive role for MFG-E8 in colorectal cancer metastasis and prognosis.

Milk fat globule epidermal growth factor 8 inhibits periodontitis in non-human primates and its gingival crevicular fluid levels can differentiate periodontal health from disease in humans.

AIM: We have previously shown that the secreted glycoprotein milk fat globule epidermal growth factor 8 (MFG-E8) has anti-inflammatory and anti-osteoclastogenic properties. Our objective was to investigate the potential of MFG-E8 as a diagnostic or therapeutic agent in periodontitis. MATERIALS AND METHODS: Periodontitis was induced in non-human primates (NHPs) by placing ligatures around posterior teeth on both halves of the mandible for a split-mouth design: one side was treated with MFG-E8-Fc and the other with Fc control. Disease was assessed by clinical periodontal examinations, radiographic analysis of bone loss, and analysis of cytokine mRNA expression in gingival biopsy samples. Gingival crevicular fluid (GCF) was collected from human healthy volunteers or subjects with gingivitis, chronic moderate periodontitis, or chronic severe periodontitis. Additionally, GCF was collected from a subset of severe periodontitis patients following scaling and root planing (SRP) and after pocket reduction surgery. GCF was analysed to quantify MFG-E8 and periodontitis-relevant cytokines using multiplex assays. RESULTS: In NHPs, sites treated with MFG-E8-Fc exhibited significantly less ligature-induced periodontal inflammation and bone loss than Fc control-treated sites. In humans, the GCF levels of MFG-E8 were significantly higher in health than in periodontitis, whereas the reverse was true for the proinflammatory cytokines tested. Consistently, MFG-E8 was elevated in GCF after both non-surgical (SRP) and surgical periodontal treatment of periodontitis patients. CONCLUSION: MFG-E8 is, in principle, a novel therapeutic agent and biomarker of periodontitis.

Serum milk fat globule epidermal growth factor 8 elevation may subdivide systemic lupus erythematosus into two pathophysiologically distinct subsets.

OBJECTIVE: Impaired clearance of apoptotic cells is a potential trigger of systemic lupus erythematosus (SLE). Milk fat globule epidermal growth factor 8 (MFG-E8) plays an important role in the clearance of dying cells. Previously, we reported serum MFG-E8 was elevated in some SLE patients. Here we further investigated the prevalence of MFG-E8 in active SLE and other autoimmune diseases and also tried to clarify the characteristics of MFG-E8-positive and -negative SLE. METHODS: Serum MFG-E8 was measured in 40 active non-treated SLE patients, 104 disease controls and 104 healthy controls by ELISA. Clinical characteristics and serum cytokine profiles were compared between MFG-E8-positive and MFG-E8-negative SLE patients. RESULTS: Prevalence of MFG-E8 was significantly higher in SLE patients (40%) than in various controls (p < 0.05). MFG-E8 level became negative after treatment, and increased again upon relapse. When compared, MFG-E8-positive SLE patients showed higher immune complex (p = 0.021) and lower complement (p = 0.004 for CH50). In contrast, MFG-E8-negative SLE patients tended to show higher CRP (p = 0.094). There was a positive correlation between MFG-E8 level and immune complex level (r s = 0.49, p = 0.049). TNF-α (p = 0.019), IFN-γ (p = 0.031) and IL-10 (p = 0.013) were significantly higher in MFG-E8-positive SLE. CONCLUSION: MFG-E8-positive SLE and -negative SLE may have different clinical features, the one with stronger immunological response and the other with stronger inflammatory response, and those two groups may be two distinct subtypes of SLE driven by different mechanisms. Further, MFG-E8 could be used as a biomarker for diagnosis and monitoring of disease activity in certain SLE patients.

MFG-E8 facilitates heart repair through M1/M2 polarization after myocardial infarction by inhibiting CaMKII.

BACKGROUND: M1/M2 macrophage polarization affects patient outcomes after myocardial infarction (MI). The relationship between milk fat globule-epidermal growth factor 8 (MFG-E8) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) on macrophage polarization after MI is unknown. To investigate the functional role of MFG-E8 in modulating cardiac M1/M2 macrophage polarization after MI, especially its influence on CaMKII signaling. METHODS: Human ventricular tissue and blood were obtained from patients with MI and controls. MFG-E8-KO mice were constructed (C57BL/6). The mice were randomized to WT-sham, sham-MFG-E8-KO, WT-PBS, rmMFG-E8 (WT injected with rmMFG-E8 10 min after MI), and MFG-E8-KO. The mouse macrophage cell line RAW264.7 was obtained. CaMKII, p-CaMKII, Akt, and NF-κB p65 were determined by qRT-PCR, western blot, and immunofluorescence. RESULTS: The MFG-E8 levels were significantly enhanced after MI in the hearts and plasma of patients with MI compared with controls. The MFG-E8 levels were significantly increased in the hearts and plasma of mice after MI. MFG-E8 was derived from cardiac fibroblasts. The administration of rmMFG-E8 improved ventricular remodeling and cardiac function after MI. rmMFG-E8 did not suppress infiltrating monocyte/macrophages into the peri-infarct area. rmMFG-E8 suppressed the polarization of macrophages to the M1 phenotype and promoted the polarization of macrophages to the M2 phenotype. rmMFG-E8 suppressed CaMKII-dependent signaling in macrophages. CONCLUSIONS: MFG-E8 and CaMKII appear to collaboratively regulate myocardial remodeling and M1/M2 macrophage polarization after MI. These observations suggest new roles for MFG-E8 in inhibiting M1 but promoting M2 macrophage polarization.

Milk fat globule epidermal growth factor 8 alleviates liver injury in severe acute pancreatitis by restoring autophagy flux and inhibiting ferroptosis in hepatocytes.

BACKGROUND: Liver injury is common in severe acute pancreatitis (SAP). Excessive autophagy often leads to an imbalance of homeostasis in hepatocytes, which induces lipid peroxidation and mitochondrial iron deposition and ultimately leads to ferroptosis. Our previous study found that milk fat globule epidermal growth factor 8 (MFG-E8) alleviates acinar cell damage during SAP via binding to αvß3/5 integrins. MFG-E8 also seems to mitigate pancreatic fibrosis via inhibiting chaperone-mediated autophagy. AIM: To speculate whether MFG-E8 could also alleviate SAP induced liver injury by restoring the abnormal autophagy flux. METHODS: SAP was induced in mice by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine or 7 hly injections of 50 µg/kg cerulein plus lipopolysaccharide. mfge8-knockout mice were used to study the effect of MFG-E8 deficiency on SAP-induced liver injury. Cilengitide, a specific αvß3/5 integrin inhibitor, was used to investigate the possible mechanism of MFG-E8. RESULTS: The results showed that MFG-E8 deficiency aggravated SAP-induced liver injury in mice, enhanced autophagy flux in hepatocyte, and worsened the degree of ferroptosis. Exogenous MFG-E8 reduced SAP-induced liver injury in a dose-dependent manner. Mechanistically, MFG-E8 mitigated excessive autophagy and inhibited ferroptosis in liver cells. Cilengitide abolished MFG-E8's beneficial effects in SAP-induced liver injury. CONCLUSION: MFG-E8 acts as an endogenous protective mediator in SAP-induced liver injury. MFG-E8 alleviates the excessive autophagy and inhibits ferroptosis in hepatocytes by binding to integrin αVß3/5.

CircSMAD2 accelerates endometrial cancer cell proliferation and metastasis by regulating the miR-1277-5p/MFGE8 axis.

OBJECTIVE: Endometrial cancer (EC) is a common gynecological malignant tumor. CircRNAs play crucial roles in cancer progression and metastasis. However, the biological functions of circRNAs in EC remain largely unknown. METHODS: CircSMAD2, miR-1277-5p, MFGE8 and relative maker protein expression in EC tissues or cell lines were analyzed by quantitative real-time polymerase chain reaction and Western blot. In vitro and in vivo functional assays, including EDU, CCK8, colony formation, transwell, tube formation and tumor xenograft assays, were conduct to explore the effects of circSMAD2 on EC. Mechanism assays were conducted to confirm the binding between miR-1277-5p and circSMAD2 or MFGE8 expression. RESULTS: Upregulation of circSMAD2 was uncovered in both EC tissues and cell lines. Functionally, silencing of circSMAD2 apparently inhibited the proliferation, migration, invasion and angiogenesis of EC cell lines in vitro. Mechanistically, circSMAD2 sponged miR-1277-5p to upregulate MFGE8 expression. The decrease of miR-1277-5p and increase of MFGE8 were observed both in EC tissues and cell lines. Then MFGE8 knockdown or miR-1277-5p upregulation suppressed EC cell oncogenic biological behavior. Rescue experiments showed that miR-1277-5p mimics countervailed the anticancer effects of circSMAD2 silencing on EC. Besides that, MFGE8 overexpression also attenuated the inhibitory action of miR-1277-5p mimic in EC. Moreover, knockdown of circSMAD2 inhibited EC growth in vivo. CONCLUSION: CircSMAD2 functions as an oncogene in promoting the progression of EC through miR-1277-5p/MFGE8 axis.

Progranulin deficiency suppresses allergic asthma and enhances efferocytosis via PPAR-γ/MFG-E8 regulation in macrophages.

Efferocytosis can resolve airway inflammation and enhance airway tolerance in allergic asthma. While previous work has reported that progranulin (PGRN) regulated macrophage efferocytosis, but it is unclear whether PGRN-mediated efferocytosis is associated with asthma. Here, we found that in an ovalbumin (OVA)-induced allergic asthma model, the airway inflammation was suppressed and the apoptosis in lung tissues was ameliorated in PGRN-deficient mice. In contrast, PGRN knockdown in human bronchial epithelial cells increased apoptosis in vitro. Furthermore, PGRN-deficient macrophages had significantly stronger efferocytosis ability than wild type (WT) macrophages both in vitro and in vivo. PGRN-deficient peritoneal macrophages (PMs) exhibited increased expression of genes associated with efferocytosis including milk fat globule-epidermal growth factor 8 (MFG-E8), peroxisome proliferator-activated receptor gamma (PPAR-γ) and sirtuin1 (SIRT1) and increased capacity to produce the anti-inflammatory mediator interleukin (IL)-10 during efferocytosis. GW9662, the inhibitor of PPAR-γ, abolished increased efferocytosis and MFG-E8 expression in PGRN-deficient PMs suggesting that PGRN deficiency enhanced MFG-E8-mediated efferocytosis through PPAR-γ. Correspondingly, efferocytosis genes were increased in the lungs of OVA-induced PGRN-deficient mice. GW9662 treatment reduced MFG-E8 expression but did not significantly affect airway inflammation. Our results demonstrated that PGRN deficiency enhanced efferocytosis via the PPAR-γ/MFG-E8 pathway and this may be one of the reasons PGRN deficiency results in inhibition of airway inflammation in allergic asthma.

Protective Effect of MFG-E8 on Necroptosis-Induced Intestinal Inflammation and Enteroendocrine Cell Function in Diabetes.

Low-grade inflammation is one of the characteristics of metabolic disorders induced by diabetes mellitus. The present study explores the underlying mechanism of milk fat globule epidermal growth factor 8 (MFG-E8) on necroptosis-induced intestinal inflammation and intestinal epithelial endocrine cell dysfunction in diabetes. Compared with the normal control group, pathological changes such as blunt and shortened villus and denuded villus tips were observed in ileum tissue of streptozotocin (STZ) induced senescence-resistant 1 (SAMR1) and senescence-accelerated prone 8 (SAMP8) diabetic mice under light microscope. Western blotting and immunohistochemistry (IHC) displayed significantly decreased protein expression of MFG-E8 in SAMR1 and SAMP8 diabetic mice, accompanied by an increased expression of phosphorylated mixed lineage kinase domain-like (p-MLKL) and HMGB1. In addition, advanced glycation end products (AGEs) significantly increased the pro-inflammatory mediators (TNF-α, IL-1ß, IL-6) and HMGB1 by activating the receptor-interacting protein kinase 3 (RIPK3)/MLKL signaling pathway in enteroendocrine STC-1 cells. D-pinitol pretreatment markedly attenuated the release of pro-inflammatory mediators and increased the expression of MFG-E8. MFG-E8 small interfering RNA (siRNA) promoted, while MFG-E8 overexpression inhibited, the activation of receptor-interacting proteins (RIPs) pathway and pro-inflammatory factors. Our study demonstrated that downregulation of MFG-E8 is an important phenomenon in the pathogenesis of diabetes-related intestinal inflammatory damage. MFG-E8 overexpression and D-pinitol intervention could protect against necroptosis-induced intestinal inflammation and maintain the function of enteroendocrine STC-1 cells in diabetes.

Chondroprotective Effects of Periodontal Ligament-Derived Stem Cells Conditioned Medium on Articular Cartilage After Impact Injury.

Paracrine factors secreted in the conditioned media (CMs) of periodontal ligament-derived stem cells (PDLSCs) have been shown to downregulate inflammatory effects of interleukin (IL)-1ß on chondrocytes wherein milk fat globule-epidermal growth factor 8 (MFG-E8) is one of the PDLSCs' highly secretory proteins. Therefore, the objective of this study was to investigate the ability of PDLSC CMs and MFG-E8 to reduce the inflammatory effects of impact injury on porcine talar articular cartilage (AC) and IL-1ß on chondrocytes, respectively. Stem cells were isolated from human periodontal ligaments. The MFG-E8 content in CM collected at 5% and 20% oxygen was measured by ELISA assay and compared across subcultures and donors. AC samples were divided into three groups: control, impact, and impact+CM. Chondrocytes were isolated from pig knees and were divided into three groups: control, IL-1ß, and IL-1ß+MFG-E8. Gene expression data were analyzed by reverse transcription-polymerase chain reaction. It was found that impact load and IL-1ß treatment upregulated IL-1ß, TNF-α, ADAMTS-4, and ADAMTS-5 gene expression in AC and chondrocytes, respectively. PDLSCs-CM prevented the upregulation of all four genes due to impact, whereas MFG-E8 prevented upregulation of IL-1ß, ADAMTS-4, and ADAMTS-5 in chondrocytes, but it did not prevent TNF-α upregulation. There were no significant differences in MFG-E8 content in CM among oxygen levels, passage numbers, or donors. The findings suggested that MFG-E8 is an effective anti-inflammatory agent contributing to the chondroprotective effects of PDLSCs-CM on acutely injured AC. Thus, introducing PDLSCs-CM to sites of acute traumatic AC injury could prevent the development of post-traumatic osteoarthritis.

Inflammatory Role of Milk Fat Globule-Epidermal Growth Factor VIII in Age-Associated Arterial Remodeling.

Background Age-associated aortic remodeling includes a marked increase in intimal medial thickness (IMT), associated with signs of inflammation. Although aortic wall milk fat globule-epidermal growth factor VIII (MFG-E8) increases with age, and is associated with aortic inflammation, it is not known whether MFG-E8 is required for the age-associated increase in aortic IMT. Here, we tested whether MFG-E8 is required for the age-associated increase in aortic IMT. Methods and Results To determine the role of MFG-E8 in the age-associated increase of IMT, we compared aortic remodeling in adult (20-week) and aged (96-week) MFG-E8 (-/-) knockout and age matched wild-type (WT) littermate mice. The average aortic IMT increased with age in the WT from 50±10 to 70±20 µm (P<0.0001) but did not significantly increase with age in MFG-E8 knockout mice. Because angiotensin II signaling is implicated as a driver of age-associated increase in IMT, we infused 30-week-old MFG-E8 knockout and age-matched littermate WT mice with angiotensin II or saline via osmotic mini-pumps to determine whether MFG-E8 is required for angiotensin II-induced aortic remodeling. (1) In WT mice, angiotensin II infusion substantially increased IMT, elastic lamina degradation, collagen deposition, and the proliferation of vascular smooth muscle cells; in contrast, these effects were significantly reduced in MFG-E8 KO mice; (2) On a molecular level, angiotensin II treatment significantly increased the activation and expression of matrix metalloproteinase type 2, transforming growth factor beta 1, and its downstream signaling molecule phosphorylated mother against decapentaplegic homolog 2, and collagen type I production in WT mice; however, in the MFG-E8 knockout mice, these molecular effects were significantly reduced; and (3) in WT mice, angiotensin II increased levels of aortic inflammatory markers phosphorylated nuclear factor-kappa beta p65, monocyte chemoattractant protein 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 molecular expression, while in contrast, these inflammatory markers did not change in knockout mice. Conclusions Thus, MFG-E8 is required for both age-associated proinflammatory aortic remodeling and also for the angiotensin II-dependent induction in younger mice of an aortic inflammatory phenotype observed in advanced age. Targeting MFG-E8 would be a novel molecular approach to curb adverse arterial remodeling.

Sphingosine 1-phosphate receptor 2 promotes erythrocyte clearance by vascular smooth muscle cells in intraplaque hemorrhage through MFG-E8 production.

Intraplaque hemorrhage (IPH) accelerates atherosclerosis progression. To scavenge excessive red blood cells (RBCs), vascular smooth muscle cells (VSMCs) with great plasticity may function as phagocytes. Here, we investigated the erythrophagocytosis function of VSMCs and possible regulations involved. Based on transcriptional microarray analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that genes up-regulated in human carotid atheroma with IPH were enriched in functions of phagocytic activities, while those down-regulated were enriched in VSMCs contraction function. Transcriptional expression of Milk fat globule-epidermal growth factor 8 (MFG-E8) was also down-regulated in atheroma with IPH. In high-fat diet-fed apolipoprotein E-deficient mice, erythrocytes were present in cells expressing VSMC markers αSMA in the brachiocephalic artery, suggesting VSMCs play a role in erythrophagocytosis. Using immunofluorescence and flow cytometry, we also found that eryptotic RBCs were bound to and internalized by VSMCs in a phosphatidylserine/MFG-E8/integrin αVß3 dependent manner in vitro. Inhibiting S1PR2 signaling with specific inhibitor JTE-013 or siRNA decreased Mfge8 expression and impaired the erythrophagocytosis of VSMCs in vitro. Partial ligation was performed in the left common carotid artery (LCA) followed by intra-intimal injection of isolated erythrocytes to observe their clearance in vivo. Interfering S1PR2 expression in VSMCs with Adeno-associated virus 9 inhibited MFG-E8 expression inside LCA plaques receiving RBCs injection and attenuated erythrocytes clearance. Erythrophagocytosis by VSMCs increased vascular endothelial growth factor-a secretion and promoted angiogenesis. The present study revealed that VSMCs act as phagocytes for RBC clearance through S1PR2 activation induced MFG-E8 release.

CX3CL1(+) Microparticles-Induced MFG-E8 Enhances Apoptotic Cell Clearance by Alveolar Macrophages.

During the resolution phase of acute lung injury, apoptotic cells release CX3CL1 as a "find-me" signal to attract alveolar macrophage transmigration toward apoptotic cells for phagocytosis. However, it is still not clear whether CX3CL1 has pro-phagocytic activity on alveolar macrophage. In this study, we investigated the role of apoptotic NB4 cells-derived CX3CL1(+) microparticles (apo-MP) on the phagocytic activity of NR8383 cells. We demonstrate that exogenous CX3CL1 and apo-MP enhanced the phagocytic activity of NR8383 cells in a CX3 CR1-dependent manner. The apo-MP-enhanced phagocytic activity on NR8383 was attenuated when apo-MP and NR8383 cells were pre-treated with anti-CX3CL1 antibodies and anti-CX3CR1 antibody, respectively, before incubating both for phagocytic assay. Further studies demonstrate that exogenous CX3CL1 and apo-MP also enhanced NR8383 cells in their surface expression and release of MFG-E8 in a CX3CR1 dependent manner. The enhanced phagocytic activity of CX3CL1-treated NR8383 cells was attenuated when NR8383 cells were pre-treated with an anti-MFG-E8 antibody before CX3CL1 treatment. We conclude that apoptotic cell-derived CX3CL1(+) microparticles enhance the phagocytic activity of NR8383 cells by up-regulating their MFG-E8 as a bridge molecule, and these contribute to the formation of phagocytic synapses between apoptotic cells and alveolar macrophages for the subsequent phagocytic clearance of apoptotic cells.

Immunological and Clinicopathological Significance of MFG-E8 Expression in Patients with Oral Squamous Cell Carcinoma.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by the activated macrophages and acts as a bridge between apoptotic cells and phagocytes. Aside from macrophages, a variety of malignant cells also express MFG-E8. The objective of this study is to elucidate the clinical relevance and significance of MFG-E8 in the tumor microenvironment (TME) of patients with oral squamous cell carcinoma (OSCC). We investigated MFG-E8 expression in 74 patients with OSCC by immunohistochemistry and evaluated the relationship between MFG-E8 expression and various clinicopathological factors including immune cell infiltration. MFG-E8 expression was detected in 34 of 74 (45.9%) patients with OSCC and a significant correlation was observed with levels of infiltrating T cells, macrophages, and immunosuppressive M2 macrophages. Furthermore, MFG-E8 expression was also associated with clinical stage, lymphatic/vascular invasion, and Ki-67+ tumor cells but not with survival. Our results suggest that MFG-E8 may play an important role in shaping the immune suppressive network in TME as well as tumor progression.

Milk Fat Globule-Epidermal Growth Factor-8 Pretreatment Attenuates Apoptosis and Inflammation via the Integrin-ß3 Pathway after Surgical Brain Injury in Rats.

Iatrogenic brain injury inevitably occurs in neurosurgical operations, leading to brain edema, ischemia, intracranial hematoma, and other postoperative complications, eventually worsening neurological outcomes of patients. If apoptotic cells are not rapidly eliminated by phagocytic engulfment, they may communicate with surrounding cells to undergo secondary necrosis and releasing toxic signals. Recent studies have shown that milk fat globule-epidermal growth factor-8 (MFGE8), which promotes phagocytosis and inhibits inflammation, is an endogenous protective factor in response to brain infarction, Alzheimer's disease, subarachnoid hemorrhage, and prion disease. In the present study, we sought to investigate the different effects of both pretreated and posttreated recombinant milk fat globule-epidermal growth factor-8 (rhMFGE8) for the surgical brain injury (SBI) rat model and potential involvement of its receptor integrin ß3 for apoptosis and neuroinflammation after SBI. One hundred and sixty-seven male rats were employed in the preset study. Experiment 1 was performed to evaluate neurological scores and MFGE8, cleaved caspase-3 (CC3), and interleukine-1 beta (IL-1ß) levels at 3, 24, and 120 h after SBI. Experiment 2 was performed to evaluate the effects of rhMFGE8 pretreatment (10 min before SBI) and rhMFGE8 posttreatment (6 h after SBI) on brain edema at 24 and 72 h after SBI. Experiment 3 was performed to evaluate the potential anti-apoptotic and anti-inflammatory effects of rhMFGE8 pretreatment and posttreatment. Experiment 4 sought to investigate the involvement of the integrin-ß3 signal in the effects of MFGE8 pretreatment. Our data showed rhMFGE8 pretreatment alleviated neurological deficits and decreased brain water content and apoptotic cells in the SBI model, which exhibited neurological dysfunction, apoptosis, and inflammation. Meanwhile, MFGE8 siRNA, which inhibited endogenous MFGE8 expression, significantly increased IL-1ß, TUNEL positive cells, and CC3. Furthermore, knockdown of its receptor integrin ß3 by siRNA abolished the effects of rhMFGE8 in the SBI model. In conclusion, we found that rhMFGE8 pretreatment effectively alleviated neurological deficits and decreased brain water content and apoptotic cells in the SBI model through the MFGE8/integrin-ß3 pathway, and treatment time was an important factor in achieving curative effects. Therefore, MFGE8 pretreatment may serve as a promising therapeutic strategy for SBI patients.