Milk Fat Globule-epidermal Growth Factor 8 Research Articles

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)-1β 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-1β 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-1β, and IL-1β+MFG-E8. Gene expression data were analyzed by reverse transcription-polymerase chain reaction. It was found that impact load and IL-1β treatment upregulated IL-1β, 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-1β, 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.

MFG-E8 Knockout Aggravated Nonalcoholic Steatohepatitis by Promoting the Activation of TLR4/NF-κB Signaling in Mice.

Nonalcoholic steatohepatitis (NASH) is the common liver disease characterized by hepatic steatosis, inflammation, and fibrosis; there are no approved drugs to treat this disease because of incomplete understanding of pathophysiological mechanisms of NASH. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a multifunctional glycoprotein, has shown anti-inflammation and antifibrosis. Here, MFG-E8 was shown to play a key role in NASH progression. Using methionine and choline deficient (MCD) diet-fed mice, we found MFG-E8 knockout exacerbated hepatic damage and steatosis as indicated by increased plasma transaminases activities and hepatic histopathologic change, higher hepatic triglycerides (TGs), and lipid accumulation. Moreover, liver fibrosis and inflammation elicited by MCD were aggravated in MFG-E8 knockout mice. Mechanistically, MFG-E8 knockout facilitated activation of hepatic toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway in MCD-fed mice. In vitro experiment, the TLR4 specific antagonist TAK-242 rescued palmitic acid- (PA-) primed lipid formation and inflammation in MFG-E8 knockout primary murine hepatocytes. These findings indicated that MFG-E8 is involved in the progression of NASH and the possible mechanism by which MFG-E8 knockout exacerbated NASH in mice is associated with activation of the TLR4/NF-κB signaling pathway.

High Levels of MFG-E8 Confer a Good Prognosis in Prostate and Renal Cancer Patients

Simple SummaryIn the present study, we analyzed the distribution and prognostic impact of milk fat globule-epidermal growth factor-8 (MFG-E8) protein expression in patients with prostate and renal cancers. Our data highlighted MFG-E8 expression by tumor cells in the epithelium. Our results also showed that low levels of MFG-E8 in prostate and renal cancers were associated with worse clinical outcomes. Furthermore, higher numbers of CD206+ cells were found in the peripheral regions of renal clear cell carcinoma that expressed lower MFG-E8 levels. Globally, our results suggest that MFG-E8 expression could potentially be used as a prognostic marker in prostate and renal cancers.Milk fat globule-epidermal growth factor-8 (MFG-E8) is a glycoprotein secreted by different cell types, including apoptotic cells and activated macrophages. MFG-E8 is highly expressed in a variety of cancers and is classically associated with tumor growth and poor patient prognosis through reprogramming of macrophages into the pro-tumoral/pro-angiogenic M2 phenotype. To date, correlations between levels of MFG-E8 and patient survival in prostate and renal cancers remain unclear. Here, we quantified MFG-E8 and CD68/CD206 expression by immunofluorescence staining in tissue microarrays constructed from renal (n = 190) and prostate (n = 274) cancer patient specimens. Percentages of MFG-E8-positive surface area were assessed in each patient core and Kaplan–Meier analyses were performed accordingly. We found that MFG-E8 was expressed more abundantly in malignant regions of prostate tissue and papillary renal cell carcinoma but was also increased in the normal adjacent regions in clear cell renal carcinoma. In addition, M2 tumor-associated macrophage staining was increased in the normal adjacent tissues compared to the malignant areas in renal cancer patients. Overall, high tissue expression of MFG-E8 was associated with less disease progression and better survival in prostate and renal cancer patients. Our observations provide new insights into tumoral MFG-E8 content and macrophage reprogramming in cancer.

Milk fat-globule epidermal growth factor 8: A potential Regulator of Cutaneous Wound Healing.

Destroying the integrity of the skin may causes disability and even death from injury or illness. Wound healing is a core mechanism to maintain skin barrier function. Milk fat-globule epidermal growth factor 8 (MFG-E8) is a key factor in wound healing and is involved in regulating blood coagulation, mediating macrophage uptake of apoptotic cells, shifting macrophages from an inflammatory to an anti-inflammatory phenotype, promoting angiogenesis, enhancing vascular endothelial growth factor (VEGF) signaling, and assisting wound tissue perfusion. However, these abilities are dysregulated in pathological conditions, such as glucose disorders and ischemic injury. Restricted application of exogenous MFG-E8 can restore function and play a beneficial role in cutaneous wound healing.

Regenerating Skeletal Muscle Compensates for the Impaired Macrophage Functions Leading to Normal Muscle Repair in Retinol Saturase Null Mice.

Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

MFG-E8 Reduces Aortic Intimal Proliferation in a Murine Model of Transplant Vasculopathy.

Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.

Milk Fat Globule Epidermal Growth Factor 8 (MFGE8) Gene Variants in Rheumatoid Arthritis and Sjögren's Syndrome.

Milk Fat Globule Epidermal Growth Factor 8 (MFGE8) deficiency and gene polymorphisms have been previously linked to systemic lupus erythematosus (SLE)-like and SLE development. Our aim was to explore whether four MFGE8 variants and MFGE8 serum levels are associated with autoimmunity susceptibility and autoimmune related atherosclerosis. DNA from 107 primary Sjogren’s syndrome (SS), 116 rheumatoid arthritis (RA) and 123 SLE patients as well as 199 HC were genotyped for the MFGE8 rs2271715, rs1878326, rs4945, rs3743388 variants by RFLP-PCR. MFGE8 serum levels were measured by ELISA. The CA genotype of rs4945 variant exhibited a protective effect against RA development, a finding not confirmed in the SS and SLE populations. The CACG haplotype exhibited a protective effect in both RA and SS patients compared to HC. Primary SS patients with IMT ≤ 0.9 mm displayed higher MGFE8 serum levels compared to those with ˃0.9 mm. Here, we report a novel association of MFGE8 variants in SS and RA susceptibility, as well as reduced MFGE8 serum levels in SS patients with heightened atherosclerotic risk.

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-1β, 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.

DCI after Aneurysmal Subarachnoid Hemorrhage Is Related to the Expression of MFG-E8.

Objective To explore the predictive value of milk fat globule epidermal growth factor 8 (MFG-E8) in the occurrence of delayed cerebral ischemia (DCI) after an aneurysmal subarachnoid hemorrhage (aSAH). Methods We recruited 32 patients with aSAH as the case group and 24 patients with unruptured aneurysms as the control group. Serum MFG-E8 levels were measured by western blot and enzyme-linked immunosorbent assay. We analyzed the relationship between MFG-E8 levels and the risk of DCI. Results The levels of serum MFG-E8 in the case group (mean = 11160.9 pg/mL) were significantly higher than those in the control group (mean = 3081.0 pg/mL, p < 0.001). MFG-E8 levels highly correlated with the World Federation of Neurosurgical Societies (WFNS) and modified Fisher scores (r = −0.691 and − 0.767, respectively, p < 0.001). In addition, MFG-E8 levels in patients with DCI (5882.7 ± 3162.4 pg/mL) were notably higher than those in patients without DCI (15818.2 ± 3771.6 pg/mL, p < 0.001). A receiver operating characteristic curve showed that the occurrence of DCI could effectively be predicted by MFG-E8 (area under the curve = 0.976, 95%CI = 0.850–1.000). Kaplan–Meier survival analysis showed a remarkable decrease in the incidence of DCI in case group individuals with high levels of MFG-E8 (≥11160.9 pg/mL, p < 0.001). Conclusion MFG-E8 may be a useful predictive marker for DCI after an aSAH and could be a promising surrogate end point.

Increased MFG-E8 at neuromuscular junctions is an exacerbating factor for sarcopenia-associated denervation.

Sarcopenia is an important health problem associated with adverse outcomes. Although the etiology of sarcopenia remains poorly understood, factors apart from muscle fibers, including humoral factors, might be involved. Here, we used cytokine antibody arrays to identify humoral factors involved in sarcopenia and found a significant increase in levels of milk fat globule epidermal growth factor 8 (MFG‐E8) in skeletal muscle of aged mice, compared with young mice. We found that the increase in MFG‐E8 protein at arterial walls and neuromuscular junctions (NMJs) in muscles of aged mice. High levels of MFG‐E8 at NMJs and an age‐related increase in arterial MFG‐E8 have also been identified in human skeletal muscle. In NMJs, MFG‐E8 is localized on the surface of terminal Schwann cells, which are important accessory cells for the maintenance of NMJs. We found that increased MFG‐E8 at NMJs precedes age‐related denervation and is more prominent in sarcopenia‐susceptible fast‐twitch than in sarcopenia‐resistant slow‐twitch muscle. Comparison between fast and slow muscles further revealed that arterial MFG‐E8 can be uncoupled from sarcopenic phenotype. A genetic deficiency in MFG‐E8 attenuated age‐related denervation of NMJs and muscle weakness, providing evidence of a pathogenic role of increased MFG‐E8. Thus, our study revealed a mechanism by which increased MFG‐E8 at NMJs leads to age‐related NMJ degeneration and suggests that targeting MFG‐E8 could be a promising therapeutic approach to prevent sarcopenia.

Endogenous neural stem cells modulate microglia and protect against demyelination.

SummaryIn response to corpus callosum (CC) demyelination, subventricular zone-derived neural progenitors (SVZdNPs) are mobilized and generate new myelinating oligodendrocytes (OLG). Here, we examine the putative immunomodulatory properties of endogenous SVZdNPs during demyelination in the cuprizone model. SVZdNP density was higher in the lateral and rostral CC regions, and demyelination was inversely correlated with activated microglial density and pro-inflammatory cytokine levels. Single-cell RNA sequencing showed that CC areas with high levels of SVZdNP mobilization were enriched in a microglial cell subpopulation with an immunomodulatory signature. We propose MFGE8 (milk fat globule-epidermal growth factor-8) and β3 integrin as a ligand/receptor pair involved in dialogue between SVZdNPs and microglia. Immature SVZdNPs mobilized to the demyelinated CC were found highly enriched in MFGE8, which promoted the phagocytosis of myelin debris in vitro. Overall, these results demonstrate that, in addition to their cell replacement capacity, endogenous progenitors have immunomodulatory properties, highlighting a new role for endogenous SVZdNPs in myelin regeneration.

MFG-E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose-Dependent Mediation of Actin Polymerization.

BackgroundMigration of vascular smooth muscle cells (VSMCs) is the main contributor to neointimal formation. The Arp2/3 (actin‐related proteins 2 and 3) complex activates actin polymerization and is involved in lamellipodia formation during VSMC migration. Milk fat globule‐epidermal growth factor 8 (MFG‐E8) is a glycoprotein expressed in VSMCs. We hypothesized that MFG‐E8 regulates VSMC migration through modulation of Arp2/3‐mediated actin polymerization.Methods and ResultsTo determine whether MFG‐E8 is essential for VSMC migration, a model of neointimal hyperplasia was induced in the common carotid artery of wild‐type and MFG‐E8 knockout mice, and the extent of neointimal formation was evaluated. Genetic deletion of MFG‐E8 in mice attenuated injury‐induced neointimal hyperplasia. Cultured VSMCs deficient in MFG‐E8 exhibited decreased cell migration. Immunofluorescence and immunoblotting revealed decreased Arp2 but not Arp3 expression in the common carotid arteries and VSMCs deficient in MFG‐E8. Exogenous administration of recombinant MFG‐E8 biphasically and dose‐dependently regulated the cultured VSMCs. At a low concentration, MFG‐E8 upregulated Arp2 expression. By contrast, MFG‐E8 at a high concentration reduced the Arp2 level and significantly attenuated actin assembly. Arp2 upregulation mediated by low‐dose MFG‐E8 was abolished by treating cultured VSMCs with β1 integrin function‐blocking antibody and Rac1 inhibitors. Moreover, treatment of the artery with a high dose of recombinant MFG‐E8 diminished injury‐induced neointimal hyperplasia and reduced VSMC migration.ConclusionsMFG‐E8 plays a critical role in VSMC migration through dose‐dependent regulation of Arp2‐mediated actin polymerization. These findings suggest that high doses of MFG‐E8 may have therapeutic potential for treating vascular occlusive diseases.

MFGE8 mitigates brain injury in a rat model of SAH by maintaining vascular endothelial integrity via TIGβ5/PI3K/CXCL12 signaling.

Leaked blood components, injured endothelial cells, local inflammatory response and vasospasm may converge to promote microthrombosis following subarachnoid hemorrhage (SAH). Previously, we showed that the milk fat globule-epidermal growth factor 8 (MFGE8) can mitigate SAH-induced microthrombosis. This present study was aimed to explore the molecular pathway participated in MFGE8-dependent protection on vascular endothelium. Immunofluorescence, immunoblot and behavioral tests were used to determine the molecular partner and signaling pathway mediating the effect of MFGE8 in vascular endothelium in rats with experimental SAH and controls, together with the applications of RNA silencing and pharmacological intervention methods. Relative to control, recombinant human MFGE8 (rhMFGE8) treatment increased 5-bromo-2'-deoxyuridine (BrdU) labeled new endothelial cells, reduced TUNUL-positive endothelial cells and elevated the expression of phosphatidylinositol 3-kinase (PI3K) and chemokine (C-X-C motif) ligand 12 (CXCL12), in the brains of SAH rats. These effects were reversed by MFGE8 RNA silencing, as well as following cilengitide and wortmannin intervention. These results suggest that MFGE8 promotes endothelial regeneration and mitigates endothelial DNA damage through the activation of the TIGβ5/PI3K/CXCL12 signaling pathway.

Propofol suppresses microglial phagocytosis through the downregulation of MFG-E8

BackgroundMicroglia are highly motile phagocytic cells in the healthy brain with surveillance and clearance functions. Although microglia have been shown to engulf cellular debris following brain insult, less is known about their phagocytic function in the absence of injury. Propofol can inhibit microglial activity, including phagocytosis. Milk fat globule epidermal growth factor 8 (MFG-E8), as a regulator of microglia, plays an essential role in the phagocytic process. However, whether MFG-E8 affects the alteration of phagocytosis by propofol remains unknown.MethodsMicroglial BV2 cells were treated with propofol, with or without MFG-E8. Phagocytosis of latex beads was evaluated by flow cytometry and immunofluorescence. MFG-E8, p-AMPK, AMPK, p-Src, and Src levels were assessed by western blot analysis. Compound C (AMPK inhibitor) and dasatinib (Src inhibitor) were applied to determine the roles of AMPK and Src in microglial phagocytosis under propofol treatment.ResultsThe phagocytic ability of microglia was significantly decreased after propofol treatment for 4 h (P < 0.05). MFG-E8 production was inhibited by propofol in a concentration- and time-dependent manner (P < 0.05). Preadministration of MFG-E8 dose-dependently (from 10 to 100 ng/ml) reversed the suppression of phagocytosis by propofol (P < 0.05). Furthermore, the decline in p-AMPK and p-Src levels induced by propofol intervention was reversed by MFG-E8 activation (P < 0.05). Administration of compound C (AMPK inhibitor) and dasatinib (Src inhibitor) to microglia blocked the trend of enhanced phagocytosis induced by MFG-E8 (P < 0.05).ConclusionsThese findings reveal the intermediate role of MFG-E8 between propofol and microglial phagocytic activity. Moreover, MFG-E8 may reverse the suppression of phagocytosis induced by propofol through the regulation of the AMPK and Src signaling pathways.

Clinical implications of milk fat globule-epidermal growth factor 8 (MFG-E8) and vitamin D levels in patients with rheumatoid arthritis and primary knee osteoarthritis

Aimof the work to determine serum levels of vitamin D and Milk fat globule-epidermal growth factor 8 (MFG-E8) in rheumatoid arthritis (RA) and primary knee osteoarthritis (OA) compared to healthy controls and their possible associations. Patients and methods: The study included 30 RA and 30 primary knee OA patients as well as 25 control. The disease activity was measured by disease activity score 28 (DAS28) in RA. The functional status was estimated by Western Ontario and McMaster Universities index (WOMAC index) in OA. While the assessment of pain was measured by the visual analog scale (VAS) in both RA and OA. Serum vitamin D and MFG-E8 levels were measured using enzyme-linked immunosorbent assay (ELISA). Results: The mean age of RA patients was 41.1 ± 9.2 years; 25 females and 5 males (F:M 5:1) and a disease duration of 6.9 ± 4.6 years. The mean age of OA patients was 48.7 ± 8.3 years; 28 females and 2 males (F:M 14:1) and disease duration was 5.4 ± 3.3 years. The mean vitamin D was significantly lower in RA (9.7 ± 5.04 ng/ml) than OA (16.4 ± 4 ng/ml) and controls (17.7 ± 3.4 ng/ml), p < 0.0001. The mean MFG-E8 serum level was significantly lower in RA and OA patients (4.9 ± 2.4 ng/mland 5.3 ± 1.2 ng/ml) than in controls (9.1 ± 3 ng/ml), p < 0.0001.Vitamin D significantly correlated with MFG-E8 (r = 0.6, p = 0.002) in RA and both inversely correlated with DAS28 (r = -0.66, p < 0.0001 and r = -0.58, p < 0.0001 respectively). Vitamin D (9.7 ng/ml) and MFG-E8 (4.9 ng/ml) significantly predicted RA (p < 0.0001) at specificity (100%) and sensitivity (66.7%). MFG-E8 at cut-off 5.3 ng/ml significantly predicted OA at specificity of 88% and sensitivity 86.7%.Conclusion: An association between low serum vitamin D and MFG-E8 levels with RA and the disease activity has been determined. Low MFG-E8 was associated with OA.

MFG-E8-derived peptide attenuates inflammation and injury after renal ischemia-reperfusion in mice

BackgroundRenal ischemia-reperfusion (renal I/R) injury may lead to acute kidney injury (AKI). After renal I/R, proinflammatory mediators cause immune cell infiltration and further injury. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) is a protein involved in cell-cell and cell-matrix interactions. MSP68 is an MFG-E8-derived peptide that inhibits neutrophil adhesion and migration. Here, we evaluated whether MSP68 attenuates renal I/R injury. Materials and methodsAdult C57BL/6 mice were subjected to bilateral renal ischemia for 30 min followed by reperfusion and intraperitoneal administration of saline (vehicle) or MSP68 (5 mg/kg). Sham animals underwent laparotomy without renal I/R. The blood collected and studied for BUN, creatinine, and LDH by colorimetry. The kidneys were analyzed for IL-6 and TNFα by qPCR, ELISA, histological injury, and apoptosis by TUNEL. ResultsAt 24 h after surgery, serum levels of BUN, creatinine, and LDH were markedly higher in vehicle-treated renal I/R mice than in sham mice, but significantly lower in MSP68-treated renal I/R mice. Similarly, compared to sham, renal levels of IL-6 mRNA and protein and TNFα protein were markedly higher in vehicle-treated renal I/R mice, but significantly lower in MSP68-treated renal I/R mice. Vehicle-treated renal I/R mice also had severe renal tubular histological injury, which was significantly lower in MSP68-treated renal I/R mice. Additionally, the kidneys of vehicle-treated renal I/R mice had a 93-fold increase in TUNEL-positive cells, which were reduced by 35% in mice treated with MSP68. ConclusionMSP68 has the potential to be developed as novel therapeutic agent for patients with AKI.

MFG-E8 attenuates inflammation in subarachnoid hemorrhage by driving microglial M2 polarization

Increasing evidence suggests that microglial polarization plays an important role in the pathological processes of neuroinflammation following subarachnoid hemorrhage (SAH). Previous studies indicated that milk fat globule-epidermal growth factor-8 (MFG-E8) has potential anti-apoptotic and anti-inflammatory effects in cerebral ischemia. However, the effects of MFG-E8 on microglial polarization have not been evaluated after SAH. Therefore, the aim of this study was to explore the role of MFG-E8 in anti-inflammation, and its effects on microglial polarization following SAH. We established the SAH model via prechiasmatic cistern blood injection in mice. Double-immunofluorescence staining, western blotting and quantitative real-time polymerase chain reaction (q-PCR) were performed to investigate the expression and cellular distribution of MFG-E8. Two different dosages (1 and 5 μg) of recombinant human MFG-E8 (rhMFG-E8) were injected intracerebroventricularly (i.c.v.) at 1 h after SAH. Brain water content, neurological scores, beam-walking score, Fluoro-Jade C (FJC), and terminal deoxynucleotidyl transferase dUTP nick endlabeling staining (TUNEL) were measured at 24 h. Suppression of MFG-E8, integrin β3 and phosphorylation of STAT3 were achieved by specific siRNAs (500 pmol/5 μl) and the STAT3 inhibitor Stattic (5 μM). The potential signaling pathways and microglial polarization were measured by immunofluorescence labeling and western blotting. SAH induction increased the levels of inflammatory mediators and the proportion of M1 cells, and caused neuronal apoptosis in mice at 24 h. Treatment with rhMFG-E8 (5 μg) remarkably decreased brain edema, improved neurological functions, reduced the levels of proinflammatory factors, and promoted the microglial to shift to M2 phenotype. However, knockdown of MFG-E8 and integrin β3 via siRNA abolished the effects of MFG-E8 on anti-inflammation and M2 phenotype polarization. The STAT3 inhibitor Stattic further clarified the role of rhMFG-E8 in microglial polarization by regulating the protein levels of the integrin β3/SOCS3/STAT3 pathway. rhMFG-E8 inhibits neuronal inflammation by transformation the microglial phenotype toward M2 and its direct protective effect on neurons after SAH, which may be mediated by modulation of the integrin β3/SOCS3/STAT3 signaling pathway, highlighting rhMFG-E8 as a potential therapeutic target for the treatment of SAH patients.

Milk Fat Globule-Epidermal Growth Factor-Factor 8 Improves Hepatic Steatosis and Inflammation.

Milk fat globule-epidermal growth factor-factor 8 (MFGE8) has been shown to be a critical extracellular molecule that mediates apoptotic signaling in the pathological process of nonalcoholic fatty liver disease (NAFLD). MFGE8 is abundantly expressed in hepatocytes, but its function in the pathogenesis of NAFLD has not been characterized. In our current study, hepatic MFGE8 showed a protective role in the pathogenesis of NAFLD. Hepatic MFGE8 deletion largely exacerbated lipid accumulation and inflammatory responses in the liver in response to overnutrition. Mechanistically, intercellular MFGE8 was shown to directly bind to apoptosis signal-regulating kinase 1 (ASK1) and to inhibit its dimerization and phosphorylation under a normal diet. However, under metabolic challenges, decreased cytoplasmic MFGE8 facilitated the dimerization and phosphorylation of ASK1 and subsequent mitogen-activated protein kinase signaling in hepatocytes. Hepatic MFGE8 is an endogenous inhibitor that halts the progression of hepatic steatosis and inflammation. Metabolic challenge-induced loss of intracellular MFGE8 facilitates ASK1 dimerization and phosphorylation. Therefore, maintaining hepatic MFGE8 levels may serve as an alternative strategy for the treatment of NAFLD.

Relationship Between Milk Fat Globule-Epidermal Growth Factor 8 and Intestinal Cytokines in Infants Born Preterm

To investigate the relationships between dietary intake and fecal concentrations of milk fat globule-epidermal growth factor 8 (MFG-E8), and between fecal concentrations of MFG-E8 and markers of intestinal inflammation in infants born preterm. Fecal samples were collected daily and enteral feedings were sampled weekly. MFG-E8 in enteral feedings and feces, and cytokine concentrations in feces were quantified by enzyme-linked immunosorbent assay. Milk MFG-E8 concentrations were significantly greater in unfortified mother's own milk (MOM) and MOM with human milk fortifier than either donor human milk or preterm formula. MFG-E8 concentrations in fecal samples were positively correlated with MFG-E8 concentrations in respective milks. High MFG-E8 exposure (≥60mL/kg/day of feedings that include MOM or MOM with human milk fortifier) was associated with lower concentrations of proinflammatory cytokines (interleukin-8, tumor necrosis factor-α, and monocyte chemoattractant protein-1) and higher concentrations of the anti-inflammatory cytokine interleukin-4 in feces, compared with low MFG-E8 exposure. Infants born preterm who were fed MOM had greater concentrations of MFG-E8 and lower concentrations of proinflammatory cytokines in fecal samples than other diets or no feedings. These data further support the protective role of MOM, possibly because of MFG-E8, against intestinal inflammation.

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

Milk fat globule epidermal growth factor (MFG-E8) is related secreted protein which links phosphatidylserine on apoptotic cells and integrin αvβ3/5 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. 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. 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). 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.