Milk Fat globule-EGF Factor Research Articles

Milk Fat Globule-EGF Factor 8 Contributes to Progression of Hepatocellular Carcinoma.

Milk fat globule-EGF factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates a wide spectrum of pathophysiological processes. MFG-E8 has been studied as a key regulator of cancer cell invasion, migration, and proliferation in different tissues and organs. However, potential roles of MFG-E8 in the growth and progression of liver cancer have not been investigated to date. Here, we analyzed 33 human hepatocellular carcinoma (HCC) samples and found that levels of MFG-E8 expression were significantly higher in HCC cells than in normal liver tissues. In addition, our in vitro gain-of-function study in three different HCC cell lines revealed that overexpression of MFG-E8 promoted the proliferation and migration of HCC cells, as determined by RT-qPCR, MTT assays, and wound healing analyses. Conversely, an MFG-E8 loss-of function study showed that proliferation capacity was significantly reduced by MFG-E8 knockdown in HCC cells. Additionally, MFG-E8 activity-neutralizing antibodies profoundly inhibited both migration and proliferation of HCC cells, attenuating their tumorigenic properties. These reductions in migration and proliferation were rescued by treatment of HCC cells with recombinant MFG-E8 protein. Furthermore, an in vivo HCC xenograft study showed that the number of proliferating HCC cells and tumor volume/weight were all significantly increased by MFG-E8 overexpression, compared to control mice. These results clearly show that MFG-E8 plays an important role in HCC progression and may provide a basis for future mechanistic studies and new strategies for the treatment of liver cancer.

A rare missense variant in the milk fat globule-EGF factor 8 (MFGE8) increases T2DM susceptibility and cardiovascular disease risk with population-specific effects.

The milk fat globule-epidermal growth factor 8 (MFGE8), also called lactadherin, is an integrin ligand and a known mediator of inflammation and atherosclerosis in T2DM in studies using animal models. However, its role in the pathophysiology of human T2DM, obesity, and cardiovascular disease has been poorly explored. Aim of thisstudy was toinvestigate the role ofa missense variant (rs371227978 C/T: Arg148His) in the MFGE8 gene identified through exome sequencing for its association with T2DM and cardiometabolic traits. Exome-wide sequencing was performed using DNA samples from 68 Sikh individuals from multi-generation pedigrees affected with diabetes onIllumina's GAIIx using "SureSelect Human All Exon" panels. We further replicated this variant by de novo genotyping in a total of 4242 individuals of the Asian Indian Diabetic Heart Study/Sikh Diabetes Study using custom TaqMan genotyping assay. We also measured circulating concentrations of Mfge8 using frozen serum aliquots by enzyme-linked immunosorbent assay. Overall, only 1.78% of 4242 individuals were carriers of this variant with MAF being 0.009. Except for the significant correlation of this variant with T2DM and triglycerides, no other quantitative risk phenotype was significant. The minor per allele-associated increased risk for T2DM showed odds ratio of 1.95 (95% CI 1.18-3.23; p = 0.008) in unadjusted model and was 1.73 (95% CI 1.02-2.93; p = 0.043) after adjusting for the age, gender, and BMI. However, there was a strong correlation between serum Mfge8 concentrations with T2DM, (r2 = 0.38; p = 0.001), fasting glucose (r2= 0.36; p = 0.002), and triglycerides (r2 = 0.33; p = 0.005). Our data revealed a significant dose-related increase in MFGE8 genotypes for affecting serum Mfge8 (p = 2.1 × 10-3) and triglyceride concentrations (p = 3.2 × 10-3). For a per riskallele-associated increase of 4.74ng/ml ± SD of 1.62ng/ml of the Mfge8 concentration was found to increase T2DM risk to 1.7 fold (95% CI from 1 to 3 fold). Here, we report for the first time a novel population-specific rare variant in the MFGE8 gene linked with the increased Mfge8 concentrations and the risk for developing T2DM and cardiovascular risk factors in a population of Punjabi Sikhs from India. In view of a strong evidence from animal studies supporting the role of Mfge8 in obesity, insulin resistance, and the development of atherosclerosis in T2DM, our findings are important and timely. If validated in a large independent dataset, early screening of Mfge8 in blood levels may especially benefit those patients with genetically elevated Mfge8 levels to preventing or reducing the risk of T2DM and cardiovascular disease.

Abstract TP279: Brain-Derived Microparticles Mediate Neuroinflammation After Stroke

Background and Purpose: Microparticles (MPs, ~ size between 0.1-1mm) are lipid encased containers, and are involved in intercellular communication and regulate inflammation. Stroke increases brain derived MP (BDMP) secretion which induces neuroinflammation. Milk fat globule-EGF factor-8 (MFGE8) promotes apoptotic cell clearance and limits pathogenic antigen cross presentation. In this study, we investigate whether BDMP affects stroke-induced neuroinflammation; whether MFGE8 treatment reduces stroke or BDMP-induced neuroinflammation and improves functional outcome after stroke. Method: 1) BDMPs were extracted from ischemic brain 24h after dMCAo by ultracentrifugation. 2) Adult (8 months) male C57BL/6J mice were subjected to dMCAo and were injected via tail vein 3h after stroke with: A) PBS (n=5/group); B) +BDMP(1.5х10 8 ,n=6/group); C)+MFGE8 (Lactadherin, 400ug/kg, n=5/group); D) +BDMP+MFGE8 (n=6/group). A battery of neurological function outcomes and immunostaining were performed. Blood plasma was used for Western blot assay. Result: 1) Compared with the Stroke+PBS control group, Stroke+BDMP significantly increases inflammatory factor expression in the circulation, increases lesion volume, neurological deficits, blood brain barrier (BBB) leakage, microglial activation, and inflammatory cell infiltration (CD45, microglia/macrophage, Neutrophils) and inflammatory factor (TNFα, IL6, IL1β) expression in brain, and increases axon/white matter (WM) damage; 2) Compared to Stroke+PBS and Stroke+BDMP groups, Stroke+MFGE8 and Stroke+BDMP+MFGE8 mice exhibited significantly improved neurological outcome; decreased lesion volume and BBB leakage, reduced axon/WM damage, and decreased inflammatory cell infiltration and inflammatory factor expression in the ischemic border, respectively. MFGE8 treatment significantly increased anti-inflammatory factor (IL10) expression in ischemic brain, and decreased IL1β expression in circulation compared to Stroke+PBS and Stroke+BDMP groups, respectively. Conclusion: BDMP increases neuroinflammation and induces worse brain damage after stroke. MFGE8 treatment reduces stroke and BDMP-induced neurological deficits possibly via its anti-inflammatory effects.

Electrostatic and Lipid Packing Effects on the Binding of Milk Fat Globule EGF Factor 8 to Phospholipid Membranes

Electrostatic and Lipid Packing Effects on the Binding of Milk Fat Globule EGF Factor 8 to Phospholipid Membranes

MFGE8 attenuates Ang-II-induced atrial fibrosis and vulnerability to atrial fibrillation through inhibition of TGF-β1/Smad2/3 pathway

Atrial fibrillation (AF) is characterized by potentiated growth of atrial fibroblasts and excessive deposition of the extracellular matrix. Atrial fibrosis has emerged as a hallmark of atrial structural remodeling linked to AF. Nonetheless, the specific mechanism underlying the progression of atrial fibrosis to AF is still largely unknown. MFGE8 (milk fat globule-EGF factor 8) is a soluble glycoprotein associated with many human diseases. Recently, a number of studies revealed that MFGE8 plays a crucial role in heart disease. Yet, MFGE8 regulation and function in the process of atrial fibrosis and vulnerability to AF remain unexplored. In this study, we found that the expression of MFGE8 was downregulated in the atriums of patients with AF compared with individuals without AF. In addition, the expression of MFGE8 was lower in atriums of angiotensin II (Ang-II)-stimulated rats as compared with the sham group. In vitro, silencing of MFGE8 by small interfering RNA significantly increased Ang-II-induced atrial fibrosis, whereas administration of recombinant human MFGE8 (rhMFGE8) attenuated the atrial fibrosis. Moreover, we found that the activated TGF-β1/Smad2/3 pathway after Ang-II treatment was significantly potentiated by the MFGE8 knockdown but inhibited by rhMFGE8 in vitro. Inhibition of integrin β3 which is the receptor for MFGE8, suppressed the TGF-β1/Smad2/3 activating effects of the MFGE8 knockdown in Ang-II-treated rat atrial fibroblasts. Finally, we administered rhMFGE8 to rats; it attenuated atrial fibrosis and remodeling and further reduced AF vulnerability induced by Ang-II, indicating that MFGE8 might have the potential both as a novel biomarker and as a therapeutic target in atrial fibrosis and AF.

The expression levels and clinical significance of MFG-E8 and CD133 in epithelial ovarian cancer

The aim of our study was to test whether there is an association between high expression of milk fat globule EGF factor 8 (MFG-E8) and CD133 presence or clinical outcomes of patients with epithelial ovarian cancer (EOC). MFG-E8 and CD133 expression levels were analyzed by immunohistochemistry in 88 EOC tumor specimens. High expression of MFG-E8 directly and significantly correlated with the presence of CD133 immunostaining (R = 0.353, p=.001), whereas immunostaining of MFG-E8 and CD133 significantly correlated with FIGO stage, tumor grade, debulking status, the dualistic model, ascites status, and nonresponse to chemotherapy (p<.05). It was also found that high expression of MFG-E8 and CD133 presence is a potent predictor of poor clinical outcomes among patients with EOC. Our study is the first to show that high expression of MFG-E8 in EOCs positively correlates with CD133 presence. Further research on MFG-E8 in EOC is needed to determine whether MFG-E8 is a new tumor marker of ovarian cancer and a new target for anticancer therapy as well as whether it can interact with cancer stem cell inhibitors for the treatment of refractory tumors.

BMPR2 dysfunction impairs insulin signaling and glucose homeostasis in cardiomyocytes.

Insulin resistance and right ventricular (RV) dysfunction are associated with lipotoxicity in heritable forms of pulmonary arterial hypertension (PAH), commonly due to mutations in bone morphogenetic protein receptor type 2 (BMPR2). How BMPR2 dysfunction in cardiomyocytes alters glucose metabolism and the response of these cells to insulin are unknown. We hypothesized that BMPR2 mutation in cardiomyocytes alters glucose-supported mitochondrial respiration and impairs cellular responses to insulin, including glucose and lipid uptake. We performed metabolic assays, immunofluorescence and Western analysis, RNA profiling, and radioactive isotope uptake studies in H9c2 cardiomyocyte cell lines with and without patient-derived BMPR2 mutations (mutant cells), with and without insulin. Unlike control cells, BMPR2 mutant cardiomyocytes have reduced metabolic plasticity as indicated by reduced mitochondrial respiration with increased mitochondrial superoxide production. These mutant cells show enhanced baseline phosphorylation of insulin-signaling protein as indicated by increased Akt, AMPK, and acetyl-CoA carboxylase phosphorylation that may negatively influence fatty acid oxidation and enhance lipid uptake, and are insulin insensitive. Furthermore, mutant cells demonstrate an increase in milk fat globule-EGF factor-8 protein (MFGE8), which influences the insulin-signaling pathway by phosphorylating AktSer473 via phosphatidylinositol 3-kinase and mammalian target of rapamycin. In conclusion, BMPR2 mutant cardiomyocytes have reduced metabolic plasticity and fail to respond to glucose. These cells have enhanced baseline insulin-signaling pattern favoring insulin resistance with failure to augment this pattern in response to insulin. BMPR2 mutation possibly blunts glucose uptake and enhances lipid uptake in these cardiomyocytes. The MFGE8-driven signaling pathway may suggest a new mechanism underlying RV lipotoxicity in PAH.

Brain-Derived Microparticles (BDMPs) Contribute to Neuroinflammation and Lactadherin Reduces BDMP Induced Neuroinflammation and Improves Outcome After Stroke.

Microparticles (MPs, ~size between 0.1 and 1 mm) are lipid encased containers derived from intact cells which contain antigen from the parent cells. MPs are involved in intercellular communication and regulate inflammation. Stroke increases secretion of brain derived MP (BDMP) which activate macrophages/microglia and induce neuroinflammation. Lactadherin (Milk fat globule–EGF factor-8) binds to anionic phospholipids and extracellular matrices, promotes apoptotic cell clearance and limits pathogenic antigen cross presentation. In this study, we investigate whether BDMP affects stroke-induced neuroinflammation and whether Lactadherin treatment reduces stroke initiated BDMP-induced neuroinflammation, thereby improving functional outcome after stroke. Middle aged (8–9 months old) male C57BL/6J mice were subjected to distal middle cerebral artery occlusion (dMCAo) stroke, and BDMPs were extracted from ischemic brain 24 h after dMCAo by ultracentrifugation. Adult male C57BL/6J mice were subjected to dMCAo and treated via tail vein injection at 3 h after stroke with: (A) +PBS (n = 5/group); (B) +BDMPs (1.5 × 108, n = 6/group); (C) +Lactadherin (400 μg/kg, n = 5/group); (D) +BDMP+Lactadherin (n = 6/group). A battery of neurological function tests were performed and mice sacrificed for immunostaining at 14 days after stroke. Blood plasma was used for Western blot assay. Our data indicate: (1) treatment of Stroke with BDMP significantly increases lesion volume, neurological deficits, blood brain barrier (BBB) leakage, microglial activation, inflammatory cell infiltration (CD45, microglia/macrophages, and neutrophils) into brain, inflammatory factor (TNFα, IL6, and IL1β) expression in brain, increases axon/white matter (WM) damage identified by decreased axon and myelin density, and increases inflammatory factor expression in the plasma when compared to PBS treated stroke mice; (2) when compared to PBS and BDMP treated stroke mice, Lactadherin and BDMP+Lactadherin treatment significantly improves neurological outcome, and decreases lesion volume, BBB leakage, axon/WM injury, inflammatory cell infiltration and inflammatory factor expression in the ischemic brain, respectively. Lactadherin treatment significantly increases anti-inflammatory factor (IL10) expression in ischemic brain and decreases IL1β expression in plasma compared to PBS and BDMP treated stroke mice, respectively. BDMP increases neuroinflammation and aggravates ischemic brain damage after stroke. Thus, Lactadherin exerts anti-inflammatory effects and improves the clearance of MPs to reduce stroke and BDMP induced neurological deficits.

Recombinant human milk fat globule-EGF factor VIII (rhMFG-E8) as a therapy for sepsis after acute exposure to alcohol

BackgroundAlcohol intake predisposes to infections and sepsis. Alcohol and sepsis inhibit the expression of milk fat globule epidermal growth factor-factor VIII (MFG-E8), a glycoprotein essential for optimal efferocytosis, resulting in the release of proinflammatory molecules and increased sepsis severity. We previously reported that recombinant mouse (rm) MFG-E8 attenuates sepsis-induced organ injury in rats with acute alcohol intoxication. In order to develop a therapy that can be safely used in humans, we have produced recombinant human (rh) MFG-E8 and evaluated its efficacy to ameliorate sepsis after acute exposure to alcohol.MethodsWe induced acute alcohol intoxication with a bolus injection of alcohol (1.75 g/kg BW) followed by an intravenous infusion of 300 mg/kg/h alcohol for 10 h. Sepsis was then induced by cecal ligation and puncture (CLP). At -10, 0, and 10 h relative to CLP, rats received MFG-E8 or vehicle (albumin) intravenously. Animals were euthanized at 20 h after CLP for blood and tissue collection. Additional groups of animals were used for a survival study.ResultsCompared to vehicle, rhMFG-E8 treatment ameliorated blood levels of proinflammatory cytokines (% improvement: TNF-α 49.8%, IL-6 34.7%) and endotoxin (61.7%), as well as of transaminases (AST 36.2%, ALT 40.1%) and lactate (18.4%). Rats treated with rhMFG-E8 also had a significant histological attenuation of the acute lung injury, as well as a reduction in the number of apoptotic cells in the thymus (43.4%) and cleaved caspase 3 (38.7%) in the spleen. In addition, rhMFG-E8 improved the 10-day sepsis survival rate from 45 to 80%ConclusionrhMFG-E8 significantly ameliorated sepsis in rats with acute alcohol exposure, demonstrating rhMFG-E8’s potential to be developed as an effective therapy for sepsis in alcohol abusers.

Bridging molecules are secreted from the skeletal muscle and potentially regulate muscle differentiation

Muscle myogenesis is an essential step for muscle development and recovery. During muscle fusion, multiple molecules are thought to be necessary for the formation of normal myotubes. Milk fat globule-EGF factor 8 (MFG-E8) and Gas6 are phosphatidylserine-recognizing bridging molecules that are secreted mainly from immune cells. In this study, we confirmed that these molecules are expressed and secreted from C2C12 cells. Mouse muscle and satellite cells also expressed these molecules. MFG-E8 was highly expressed and secreted in both undifferentiated and differentiated C2C12 cells. We observed that MFG-E8 and Gas6 were bound to the surface of differentiated C2C12 cells more compared with undifferentiated cells.Additionally, the treatment of recombinant MFG-E8 upregulated expression of myogenic genes and suppressed apoptosis during myogenesis in C2C12 cells. In this paper, we discuss the presence of novel functional molecules expressed and secreted in the skeletal muscle. The results of this study suggest that bridging molecules are one of the determinants of myogenesis or other muscle responses.

Improvement of Insect Tea Extract on D-galactose Induced Aging in Mice

Milk fat globule-EGF factor 8 (MFG-E8) is one of the major proteins in milk fat globule membrane. In this study, mouse-derived C2C12 myoblast cells were served as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocytes formation comprehensively, a liquid chromatography coupled with tandem mass spectrometry label-free semi-quantitative approach was used to analyse the myogenic C2C12 proliferation program. Over 1987 proteins were identified in C2C12 cells. The MFG-E8 (200 μg/mL) and MFG-E8 (500 μg/mL) with significant differences were compared based on the relative abundance. The result profiles of regulation of MFG-E8 to the expression of proteins in C2C12 cells revealed that differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across during the C2C12 cell proliferation process. Based on the analysis of KEGG and STRING database, further to verification the expression of PI3K and ERK phosphorylation levels by Western blot. This study found that the data of proteomic was complementary to recent MFG-E8 studies of protein expression patterns in developing myotubes and provided a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.

Serum milk fat globule-EGF factor 8 (MFG-E8) as a diagnostic and prognostic biomarker in patients with hepatocellular carcinoma

Current serum hepatocellular carcinoma (HCC) biomarkers are insufficient for early diagnosis. We aimed to clarify whether serum MFG-E8 can serve as a diagnostic or prognostic biomarker of HCC. Serum MFG-E8 levels of 282 HCC patients, who underwent primary hepatectomy, were examined by ELISA. We also quantified serum MFG-E8 levels in patients with chronic hepatitis (CH), liver cirrhosis (LC), as well as in healthy volunteers (HVs). Serum MFG-E8 levels were significantly lower in HCC patients than in HVs regardless of the etiology of liver disease (3.6 ± 0.1 vs 5.8 ± 0.2 ng/mL, p < 0.0001), and recovered after treatment of HCC. Serum MFG-E8 levels in CH and LC patients were comparable to those in HVs. Serum MFG-E8 could detect HCCs, even α-fetoprotein (AFP)-negative or des-γ-carboxy prothrombin (DCP)-negative HCCs, in CH and LC patients. Our new HCC prediction model using MFG-E8 and DCP (Logit(p) = 2.619 − 0.809 × serum MFG-E8 + 0.0226 × serum DCP) distinguished HCC patients from CH and LC patients with an area under the curve of 0.923, a sensitivity of 81.1%, and a specificity of 89.8%. Futhermore, low preoperative serum MFG-E8 was an independent predictor of poor overall survival. Thus, serum MFG-E8 could serve as a feasible diagnostic and prognostic biomarker for HCC.

Discovery of crucial cytokines associated with abdominal aortic aneurysm formation by protein array analysis.

As a common disease, abdominal aortic aneurysm (AAA) features permanently progressively dilated abdominal aorta. Various cytokines are implicated in AAA pathogenesis. Clarification of involved cytokines combined with functional analysis may provide new insights into AAA pathogenesis. Using a mouse model, this study analyzed the cytokine profiles in AAA. Cytokines were measured in AAA tissues of saline control or angiotensin II-treated ApoE−/− mice using an antibody array of 200 cytokines, cytokine receptors, and related proteins. Statistical analysis revealed that 21 of 200 proteins were differentially expressed in AAA. These differentially expressed proteins were subjected to function and pathway enrichment analysis, which revealed that leukocyte migration and positive regulation of cell adhesion were the most significant biological processes. Specific signaling pathways, including Janus kinase/signal transducers and activators of transcription and cytokine–cytokine receptor interaction, were prominent in Kyoto encyclopedia of genes and genomes pathway enrichment analysis. Importantly, our data identified cytokines which had not previously been illustrated in AAA pathogenic pathways. Bivariate correlation analysis between these cytokines and protease activity showed that granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein 1 g, cardiotrophin 1, milk fat globule-EGF factor 8 protein, interleukin 33, and periostin were positively correlated with matrix metalloprotease 1 (MMP-1), MMP-9, cathepsin B, and cathepsin L. G-CSF was positively correlated with cathepsin L. In conclusion, these results demonstrate that cytokine profile is significantly altered in AAA, and that the newly identified crucial cytokines may function potentially in AAA pathogenesis. Impact statement Various cytokines are known contributors to abdominal aortic aneurysm (AAA) pathologic processes, but the mechanisms underlying the pathogenesis remains unclear. We illustrated the altered cytokine profiles in AAA by high throughput antibody array of 200 cytokines, cytokine receptors and related proteins, as well as bioinformatics analysis of differentially expressed proteins in lesion tissues from AAA mice infused with angiotensin II. Functional analyses of differentially expressed cytokines showed clustering on cell migration and adhesion processes. More importantly, crucial cytokines whose association with AAA formation had not been established were identified. Significant correlations were found between these cytokines and protease activity. This study identifies several crucial markers for further researches on the molecular basis of AAA.

The glycoproteins EDIL3 and MFGE8 regulate vesicle-mediated eggshell calcification in a new model for avian biomineralization

The avian eggshell is a critical physical barrier, which permits extra-uterine development of the embryo. Its formation involves the fastest known biomineralization process in vertebrates. The eggshell consists of proteins and proteoglycans that interact with the mineral phase to impart its specific microstructure and mechanical properties. In this study, we investigated the role of epidermal growth factor (EGF)-like repeats and discoidin-like domains 3 (EDIL3) and milk fat globule-EGF factor 8 (MFGE8), two glycoproteins that are consistently detected in eggshell proteomes. We verified their common evolutionary history and identified the timing of the duplication event giving rise to these two distinct proteins. Edil3/mfge8 chromosomal locations revealed a nested syntenous relationship with other genes (hapln1/hapln3 and vcan/acan) that are also involved in vertebrate calcification. EDIL3 and MFGE8 proteins possess EGF-like and coagulation factor 5/8 (F5/8C) domains, and their 3D structures predicted that they bind calcium and extracellular vesicles. In chicken, we confirmed the presence of EDIL3 and MFGE8 proteins in eggshell, uterine fluid, and uterus. We observed that only edil3 is overexpressed in tissues in which eggshell mineralization takes place and that this overexpression occurs only at the onset of shell calcification. We therefore propose a model in which EDIL3 and, to a lesser extent, MFGE8 proteins guide vesicles containing amorphous calcium carbonate to the mineralization site. This model was supported by the observation that extracellular vesicles accumulate in uterine fluid during eggshell calcification and that they contain high levels of calcium, carbon, and oxygen that correspond to calcium carbonate.

Abstract 247: Phagocytosis-mediated Activation of Smad3 in Macrophages Mediates Anti-inflammatory Transition and Protects From Adverse Post-infarction Remodeling

Macrophages are essential regulators in tissue injury, repair and fibrosis. Although activation of TGF-β signaling cascades critically modulates macrophage phenotype in vitro, the in vivo role of macrophage TGF-β signaling in homeostasis and disease is unknown. Our study examined the role of macrophage-specific TGF-β/Smad3 signaling in a mouse model of myocardial infarction (MI). TGF-βs markedly activated Smad3 in isolated macrophages, without affecting Smad-independent pathways. Infarct macrophages, but not neutrophils exhibited Smad3 activation. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-β1 release. Myeloid cell-specific Smad3 knockout mice (MyS3KO) had no baseline defects, but exhibited increased late mortality and accentuated remodeling following MI. Adverse outcome in MyS3KO mice after MI was associated with perturbations in phagocytic activity, with defective transition of macrophages to an anti-inflammatory phenotype and with scar expansion. Milk-fat globule EGF factor-8 (Mfge8) partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Perturbed anti-inflammatory transition of Smad3 null macrophages was associated with marked attenuation of phagocytosis-mediated PPAR induction. We demonstrate that Smad3 is activated in phagocytic macrophages, critically regulates their function and mediates anti-inflammatory transition, and protects the infarcted heart from adverse remodeling.

Idiopathic degenerative thoracic aneurysms are associated with increased aortic medial amyloid

Objective: To explore the relationship of aortic medial amyloid with biochemical and micromechanical properties of the aortic wall in aneurysm patients.Methods: Human aortic tissues removed during aneurysm surgery from tricuspid (idiopathic degenerative aneurysm, DA) and bicuspid valve (BAV) patients were subjected to oscillatory nanoindentation experiments to determine localised mechanical properties of the tissue (shear storage modulus, G´ and shear loss modulus, G˝). Collagen, elastin, matrix metalloproteinase 2 and glycosaminoglycans concentrations were determined, along with relative levels of aortic medial amyloid-related factors (medin, milk fat globule-EGF factor 8, oligomers and fibrils). Measurements were combined with clinical data and statistical analyses performed.Results: The DA cohort can be divided based on their phenotype. One group shared similar characteristics with BAV patients, termed bicuspid like phenotype-tricuspid valve. The second group had high amyloid oligomer species present with a significantly lower G´ (p = .01), indicative of reduced elastic response of the tissue, termed amyloid-rich.Conclusions: We identified a group of DA patients with high amyloid oligomers and altered micromechanical and structural properties of the vessel wall. We propose these findings as a cause for aneurysm formation in these patients. Amyloid is not found in BAV patients, suggesting at least two distinct mechanisms for pathogenesis.

Microbiota metabolites SCFA stimulate epithelial migration to promote wound healing through MFGE8 and PAK1

Abstract How microbiota regulate intestinal homeostasis is still not completely understood. Emerging evidence indicates that gut microbiota metabolites play crucial roles in maintenance of intestinal homeostasis and regulation of Inflammatory Bowel Disease (IBD). Many patients with IBD are deficient in the microbial metabolites of dietary fibers, short chain fatty acids (SCFAs). To date, the evidence for use of SCFAs as a treatment for IBD has been inconsistent, highlighting the need for further investigation. In this study, we demonstrate that SCFAs promote intestinal epithelial cell (IEC) wound healing. Treatment of mouse and human IEC in vitro with the SCFAs acetate, propionate, and butyrate enhanced wound closure by promoting cell migration, which was independent of cell proliferation. Further analysis of random cell migration revealed that SCFAs increased cell speed, cell persistence, and consequently mean squared displacement. To investigate the mechanism of enhanced migration, we performed microarray of mouse-derived enteroids, which identified p21 activated kinase 1 (PAK1) and milk fat globule-EGF factor 8 (MFGE8), two genes implicated in tissue repair and motility, to be highly upregulated. We confirmed upregulation of MFGE8 and PAK1 via qRT-PCR and western blot. Inhibition of PAK1 and CRISPR knockout of MFGE8 abrogated the effects of SCFAs on migration. Lastly, the above effects were dependent upon SCFAs inhibition of histone deacetylases (HDAC), as treatment of IECs with the HDAC inhibitor, trichostatin A, fully recapitulated the effects of SCFAs on migration and expression of MFGE8 and PAK1. These findings elucidate a pathway in which SCFAs inhibition of histone deacetylases may enhance wound healing in IBD.

MFGE8 protects against CCl4 -induced liver injury by reducing apoptosis and promoting proliferation of hepatocytes.

Milk fat globule-EGF factor 8 (MFGE8) has been reported to play various roles in acute injury and inflammation response. However, the role of MFGE8 in liver injury is poorly investigated. The present research was designed to clarify the expression and function of MFGE8 in carbon tetrachloride (CCl4 )-induced liver injury. Using serum cytokine arrays, we selected a promising cytokine MFGE8 as the candidate in the process of hepatitis-fibrosis-hepatocellular carcinoma (HCC) progression, based on the elevated expression in both hepatic fibrosis and HCC models. We validated the increased expression of MFGE8 in liver tissues and serum samples of acute and chronic CCl4 -induced mice. Immunohistochemistry staining of mouse liver tissues indicated that elevated MFGE8 expression was mainly derived from the injured hepatocytes. In addition, MFGE8 expression in the supernatant of primary hepatocytes was accumulated with prolongation of culture time, and CCl4 treatment further increased the expression of MFGE8. Moreover, a strong correlation between serum MFGE8 expression and liver transaminase activities suggested that MFGE8 may be a novel candidate in liver injury. Intriguingly, mice pretreated with MFGE8 were protected from CCl4 -induced liver injury through antiapoptosis role in the early stage and proproliferation role in the late stage. MFGE8 reduced apoptosis by inhibiting the activation of IRE1α/ASK1/JNK pathway and promoted proliferation by phosphorylation of ERK and AKT. Moreover, serum MFGE8 expression was increased in hepatitis patients while decreased in liver cirrhosis patients. All the results suggest MFGE8 as a novel marker and promising therapeutic agent of liver injury.

Reduction in milk fat globule-EGF factor 8 inhibits triple-negative breast cancer cell viability and migration.

Milk fat globule-EGF factor 8 (MFG-E8) has been demonstrated to be associated with the progression and metastasis of breast cancer, although the underlying mechanisms remain undefined. The aim of the present study was to explore the role of MFG-E8 in human breast cancer and examine the underlying molecular mechanisms. Reverse transcription-quantitative polymerase chain reaction analysis was used to evaluate the expression levels of MFG-E8 in human breast carcinoma cell lines. An MFG-E8 small interfering RNA lentiviral vector was constructed and transfected into MDA-MB-231 cells. The results indicated that the in vitro silencing of MFG-E8 significantly inhibited the viability, invasion and migration of breast cancer cells. By using a flow cytometric approach, the knockdown of MFG-E8 was revealed to significantly induce cell cycle arrest at the G2/M phase and cell apoptosis. Furthermore, the downregulation of MFG-E8 induced the activation of apoptosis-associated proteins, and inhibited the expression of matrix metalloproteinase and epithelial-mesenchymal transition-associated proteins. Collectively, the results of the present study emphasize the importance of MFG-E8 deregulation in mammary carcinogenesis and its potential use as a biomarker for the diagnosis of breast carcinomas.

MFG-E8 overexpression is associated with poor prognosis in breast cancer patients

BackgroundMFG-E8(Milk fat globule-EGF factor 8), a secreted glycoprotein, plays an exceptional role in various diseases. MFG-E8 overexpression is found in a variety of cancers. However, it remains unclear whether MFG-E8 overexpression is associated with the clinicopathological characteristics and prognosis of human breast cancer. Materials and methodsIn this study, we detected the expression and localization of MFG-E8 protein in breast cancer and cancer-adjacent tissues using immunohistochemical staining, Western blot analysis and immunofluorescence. We analyzed the association between MFG-E8 expression and clinical characteristics and outcomes of breast cancer patients with different HR and HER2 statuses. ResultsOur results confirmed that MFG-E8 expression increased significantly in breast cancer compared with cancer-adjacent tissues by immunohistochemical staining (P < 0.001). Similarly, the Western blot results further confirmed the increased expression of MFG-E8 in breast cancer compared with cancer-adjacent tissues (P = 0.001). Immunofluorescence staining showed that MFG-E8 was mainly localized in the cytoplasm and membrane of tumor cells, consistent with the immunohistochemical staining results. The high expression levels of MFG-E8 showed a greater association with lymph node metastasis, TNM stage and histological grade (P < 0.001). Moreover, high MFG-E8 expression was related to a shortened overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001). Bioinformatics analysis with a Kaplan-Meier plotter also demonstrated a strong association of MFG-E8 mRNA overexpression with a short OS and DFS compared with low MFG-E8 expression (P = 0.040, P = 0.005). ConclusionsOur findings indicate that MFG-E8 may be a potential marker for poor prognosis and survival in breast cancer.