Fat Globule Epidermal Growth Factor Research Articles

Acetylation-ubiquitination crosstalk of DJ-1 mediates microcalcification formation in diabetic plaques via collagen-matrix vesicles interaction.

Microcalcification increases the vulnerability of plaques and has become an important driver of acute cardiovascular events in diabetic patients. However, the regulatory mechanisms remain unclear. DJ-1, a multifunctional protein, may play a potential role in the development of diabetic complications. Therefore, this study aims to explore the relationship between DJ-1 and microcalcification in diabetic plaques and investigate the mechanisms. The regulatory relationship between DJ-1 and diabetic vascular microcalcification was determined in anterior tibial arteries from diabetic foot amputated patients, a diabetic apolipoprotein E-deficient (ApoE-/-) mouse model, and a vascular smooth muscle cell (VSMC) model. The ubiquitination and acetylation levels of DJ-1 were detected, and the acetylation-ubiquitination crosstalk was explored. Then, the regulatory effects of DJ-1 on receptor for advanced glycation end products (RAGE) were clarified. Further, the role of DJ-1 in collagen- matrix vesicles (MVs) interaction in diabetic microenvironment was observed. The collagen interacting surface protein of MVs was verified with proteomics and the biomimetic MVs model.In clinical samples, the number of microcalcification nodules in anterior tibial artery plaques was negatively correlated with DJ-1 expression. In diabetic ApoE-/- mice and VSMCs models, knocking down DJ-1 significantly increased the number of microcalcified nodules. N-acetyltransferase 10 (NAT10) was an acetyltransferase of DJ-1. NAT10 could crosstalk the ubiquitination of DJ-1 and enhance the ubiquitination of DJ-1 by E3 ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). Besides, the knockdown of DJ-1 activated signal transducer and activator of transcription 1 (STAT1), and then STAT1 could bind to RAGE promoter, thus upregulating RAGE. Furthermore, the knockdown of DJ-1 significantly promoted collagen-MVs interaction in diabetic microenvironment. Milk fat globule epidermal growth factor 8 (MFGE8) may serve as a collagen-interacting protein. The coating of MFGE8 protein could increase the interaction between collagen and biomimetic MVs. In the diabetic microenvironment, DJ-1 was a protective factor for vascular microcalcification. NAT10- and TRIM32-mediated acetylation-ubiquitination crosstalk resulted in the degradation of DJ-1. The decrease of DJ-1 could activate DJ-1/STAT1/RAGE microcalcification signal. Further, under the stimulation of DJ-1-mediated microcalcification signal, VSMCs released MVs with high abundance of MFGE8. MFGE8 promoted collagen-MVs interaction and finally accelerated the formation of microcalcification.

Potentiating microglial efferocytosis by MFG‐E8 improves survival and neurological outcome after successful cardiopulmonary resuscitation in mice

AbstractBrain injury represents the leading cause of mortality and disability after cardiopulmonary resuscitation (CPR) from cardiac arrest (CA), in which the accumulation of dying cells aggravate tissue injury by releasing proinflammatory intracellular components. Microglia play an essential role in maintaining brain homeostasis via milk fat globule epidermal growth factor 8 (MFG‐E8)‐opsonized efferocytosis, the engulfment of dying cells and debris. This study investigates whether potentiating microglia efferocytosis by MFG‐E8 provides neuroprotection after CA/CPR. After 8‐minute asphyxial CA/CPR, male adult C57BL/6J mice were randomly assigned to receive recombinant mouse MFG‐E8 (rmMFG‐E8) or vehicle. We evaluated the survival and neurological deficits of mice, along with histological damages, phagocytosis index of dying cells, and microglia polarization. A transcriptome analysis was conducted to explore the downstream molecules modulated by MFG‐E8. In mice resuscitated from CA, rmMFG‐E8 administration significantly enhanced the efferocytosis of apoptotic cells by microglia, improved the survival and neurological function of mice, and attenuated neuropathological injuries. Additionally, rmMFG‐E8 induced a prominent alteration in microglial gene expression and promoted a shift from a proinflammatory phenotype to an anti‐inflammatory phenotype. Moreover, rmMFG‐E8 treatment induced up‐regulation of interferon regulatory factor 7 (IRF7), and IRF7 gene silencing largely reversed the neuroprotective effects of rmMFG‐E8. This study demonstrates that rmMFG‐E8 improves survival and neurological outcomes after CA/CPR by enhancing microglial efferocytosis and reshaping the inflammatory microenvironment in brain tissue. Potentiating MFG‐E8 is a promising strategy to combat post‐CA brain injury.

Linoleic Acid Enhances Renal Tubular Epithelial Cells Autophagy Caused by Calcium Oxalate Monohydrate Crystals.

High intake of dietary linoleic acid may increase the incidence of many diseases. The aim of this research is to examine the impact of linoleic acid on the damage caused by calcium oxalate kidney stones on renal tubular epithelial cells. Calcium oxalate monohydrate (COM) crystals were prepared and used to treat HK-2 cells, which were further treated with different concentrations of linoleic acid invitro. Also, a small-interfering RNA lentiviral vector of milk fat globule epidermal growth factor 8 (MFGE8) was constructed and transfected into HK-2 cells. The cell viability, level of intracellular ROS and autophagy were tested. Invivo experiments were also carried out with a rat model for renal urolithiasis treated with linoleic acid. The results indicated that COM crystals promoted crystal deposition and apoptosis, increased levels of intracellular Ca2+ and ROS levels and inhibited the proliferation of HK-2 cells. Linoleic acid exacerbated the damage of COM crystal-treated HK-2 cells and renal tubular epithelial cells of the rat model for renal urolithiasis, which can be partially reversed by downregulation of MFGE8. These results collectively suggest that linoleic acid might enhance the damage of renal tubular epithelial cells caused by COM crystals.

MFG-E8 Ameliorates Nerve Injury-Induced Neuropathic Pain by Regulating Microglial Polarization and Neuroinflammation via Integrin β3/SOCS3/STAT3 Pathway in Mice.

Spinal microglial polarization plays a crucial role in the pathological processes of neuropathic pain following peripheral nerve injury. Accumulating evidence suggests that milk fat globule epidermal growth factor-8 (MFG-E8) exhibits anti-inflammatory effect and regulates microglial polarization through the integrin β3 receptor. However, the impact of MFG-E8 on microglial polarization in the context of neuropathic pain has not yet been investigated. In this study, we evaluated the effect of MFG-E8 on pain hypersensitivity and spinal microglial polarization following spared nerve injury (SNI) of the sciatic nerve in mice. We determined the molecular mechanisms underlying the effects of MFG-E8 on pain hypersensitivity and spinal microglial polarization using pain behavior assessment, western blot (WB) analysis, immunofluorescence (IF) staining, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and small interfering RNA (siRNA) transfection. Our findings indicate that SNI significantly increased the levels of MFG-E8 and integrin β3 expressed in microglia within the spinal cord of mice. Additionally, we observed that intrathecal injection of recombinant human MFG-E8 (rhMFG-E8) alleviated SNI induced-mechanical allodynia and thermal hyperalgesia. Furthermore, the results suggested that rhMFG-E8 facilitated M2 microglial polarization and ameliorated neuroinflammation via integrin β3/SOCS3/STAT3 pathway in the spinal cord of mice with SNI. Importantly, these effects were negated by integrin β3 siRNA, or SOCS3 siRNA. These results demonstrate that MFG-E8 ameliorates peripheral nerve injury induced-mechanical allodynia and thermal hyperalgesia by driving M2 microglial polarization and mitigating neuroinflammation mediated by integrin β3/SOCS3/STAT3 pathway in the spinal cord of mice. MFG-E8 may serve as a promising target for the treatment of neuropathic pain.

MFGE8 promotes adult hippocampal neurogenesis in rats following experimental subarachnoid hemorrhage via modifying the integrin β3/Akt signaling pathway

Subarachnoid hemorrhage (SAH) is one of the most severe type of cerebral strokes, which can cause multiple cellular changes in the brain leading to neuronal injury and neurological deficits. Specifically, SAH can impair adult neurogenesis in the hippocampal dentate gyrus, thus may affecting poststroke neurological and cognitive recovery. Here, we identified a non-canonical role of milk fat globule epidermal growth factor 8 (MFGE8) in rat brain after experimental SAH, involving a stimulation on adult hippocampal neurogenesis(AHN). Experimental SAH was induced in Sprague-Dawley rats via endovascular perforation, with the in vivo effect of MFGE8 evaluated via the application of recombinant human MFGE8 (rhMFGE8) along with pharmacological interventions, as determined by hemorrhagic grading, neurobehavioral test, and histological and biochemical analyses of neurogenesis related markers. Results: Levels of the endogenous hippocampal MFGE8 protein, integrin-β3 and protein kinase B (p-Akt) were elevated in the SAH relative to control groups, while that of hippocalcin (HPCA) and cyclin D1 showed the opposite change. Intraventricular rhMGFE8 infusion reversed the decrease in doublecortin (DCX) immature neurons in the DG after SAH, along with improved the short/long term neurobehavioral scores. rhMGFE8 treatment elevated the levels of phosphatidylinositol 3-kinase (PI3K), p-Akt, mammalian target of rapamycin (mTOR), CyclinD1, HPCA and DCX in hippocampal lysates, but not that of integrin β3 and Akt, at 24 hr after SAH. Treatment of integrin β3 siRNA, the PI3K selective inhibitor ly294002 or Akt selective inhibitor MK2206 abolished the effects of rhMGFE8 after SAH. In conclusion, MFGE8 is upregulated in the hippocampus in adult rats with reduced granule cell genesis. rhMFGE8 administration can rescue this impaired adult neurogenesis and improve neurobehavioral recovery. Mechanistically, the effect of MFGE8 on hippocampal adult neurogenesis is mediated by the activation of integrin β3/Akt pathway. These findings suggest that exogenous MFGE8 may be of potential therapeutic value in SAH management.Graphical abstract and proposed pathway of rhMFGE8 administration attenuate hippocampal injury by improving neurogenesis in SAH models. SAH caused hippocampal injury and neurogenesis interruption. Administered exogenous MFGE8, recombinant human MFGE8(rhMFGE8), could ameliorate hippocampal injury and improve neurological functions after SAH. Mechanistically, MFGE8 bind to the receptor integrin β3, which activated the PI3K/Akt pathway to increase the mTOR expression, and further promote the expression of cyclin D1, HPCA and DCX. rhMFGE8 could attenuated hippocampal injury by improving neurogenesis after SAH, however, know down integrin β3 or pharmacological inhibited PI3K/Akt by ly294002 or MK2206 reversed the neuro-protective effect of rhMFGE8.

Milk Fat Globule-EGF Factor 8 (MFGE8) Mitigates Cognitive Impairment in Rats with Sepsis-Associated Encephalopathy: An fMRI Study.

Sepsis-associated encephalopathy (SAE) impairs hippocampal microglial efferocytosis, causing cognitive deficits. Previous research found that milk fat globule epidermal growth factor 8 protein (MFGE8) stimulates efferocytosis, reducing hippocampal inflammation in SAE rats. In this study, we explore MFGE8's role in alleviating cognitive impairment and its impact on neural activity using functional magnetic resonance imaging (fMRI). Sixty male Sprague Dawley rats were divided into four groups: Sham, cecal ligation and puncture (CLP), CLP+MFGE8, and CLP+MFGE8+CGT (Cilengitide). After CLP, CLP+MFGE8 rats received intracerebroventricular MFGE8 (3.3 µg), while CLP+MFGE8+CGT rats received intraperitoneal Cilengitide (10 mg/kg). We assessed cognitive function with the Morris water maze and open field test over five days. Eight days post-surgery, rats underwent T2-weighted magnetic resonance imaging (MRI) and resting state (rs)-fMRI scans. Brain tissues were collected for western blot, hematoxylin-eosin (HE) staining, and immunofluorescence. Statistical analysis employed one-way analysis of variance (ANOVA) followed by Tukey's post-test for multiple comparisons. MFGE8 improved neurobehavioral performance in open field task (OFT) and morris water maze (MWM) tests. fMRI indicated a significant reduction in abnormal neural activity in the right hippocampal CA1, CA3, and dentate gyrus of SAE rats following MFGE8 treatment. Voxel-based morphometry (VBM) analysis revealed decreased high-signal areas in the hippocampus, along with reduced hippocampal volume due to alleviated neural edema. Western blot analysis demonstrated that MFGE8 enhanced ras-related C3 botulinum toxin substrate 1 (Rac1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) expression in the rat hippocampus, while CGT reduced these protein levels. Behavioral experiments and fMRI results confirmed that CGT reversed the cognitive effects of MFGE8 by inhibiting microglial αVβ3/αVβ5 integrin receptors. Our findings show that MFGE8 reduced amplitude of low-frequency fluctuations (ALFF) values in the right hippocampal CA1, CA3, and the dentate gyrus, mitigating abnormal neural activity and decreasing hippocampal volume. This led to an improvement in cognitive dysfunction in SAE rats. These results suggest that MFGE8 enhances microglial efferocytosis by activating αVβ3 and αVβ5 integrin receptors on microglial surfaces, ultimately improving cognitive function in SAE rats.

Ex vivo MFG-E8 treatment improves the function of lungs procured from cardiac death donors in preclinical porcine model

Ex vivo lung perfusion (EVLP) is a promising technology that allows the re-evaluation of donor lungs and has the potential to improve marginal lung reconditioning. The present study focused on the effects of milk fat globule epidermal growth factor 8 (MFG‐E8) on the function of donation after circulatory death (DCD) lungs during EVLP and transplant reperfusion. Domestic swine were assigned to 4 groups. In the control group, the donor lungs lacking warm ischemia were preserved in Perfadex for 4 h. The swine in the other three groups underwent hypoxic arrest, followed by 1 h of warm ischemia. The DCD lungs were procured and randomly divided into three groups: cold static preservation (DCD-CSP) group, DCD-EVLP group, and DCD-MFG-E8 group. The left lung of all groups was transplanted and reperfused. During EVLP and reperfusion, lung functions and pathological evaluations were performed. Treatment with MFG-E8 resulted in significantly improved blood oxygenation. The mean pulmonary artery pressure, peak airway pressure, and expression of IL-1β, IL-6, and IL-12 were significantly lower but IL-10 was higher in the DCD -MFG-E8 group. Furthermore, the lung injury severity score, pulmonary edema, and wet-to-dry weight ratio were also reduced in MFG-E8-treated lungs. However, the pulmonary vascular resistance and expression of TNF-α did not differ from the DCD -EVLP group but were significantly lower than in the DCD -CSP group. Adding MFG-E8 into the perfusate during EVLP obtains optimal graft function of lungs from DCD. This finding, if confirmed clinically, can be applied to recondition grafts and expanded use of DCD lungs.

Characterization and evaluation of MFG-E8 protein-derived peptides: From isolation to functional assessment in skeletal muscle lipid metabolism

This research investigated preparation, identification and relative properties of milk fat globule epidermal growth factor Ⅷ (MFG-E8) protein-derived peptides. MFG-E8 protein was extracted from bovine milk and peptides were isolated and obtained using an in vitro simulating gastrointestinal digestion model, the peptide sequence was then identified. High-abundance MFG-E8 peptides bind well to the protein domain of pancreatic lipase was selected. Enzyme kinetic studies exhibited that the inhibition effect of KDPG and YAR on pancreatic lipase activity was reversible and non-competitive inhibition. Fluorescence spectroscopic analysis showed that KDPG and YAR acted as fluorescence quenchers of pancreatic lipase and exhibited certain dose-dependent effect. In vivo animal experiments further verified the regulatory effects of MFG-E8 peptides on lipid metabolism and skeletal muscle profiles, which provided the potential role of KDPG and YAR in regulating lipid metabolism and alleviating skeletal muscle dysfunction via AMPK/SREBP-1/SCD-1 signaling pathway.

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

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 αvβ3/5 integrins. MFG-E8 also seems to mitigate pancreatic fibrosis via inhibiting chaperone-mediated autophagy. To speculate whether MFG-E8 could also alleviate SAP induced liver injury by restoring the abnormal autophagy flux. 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 αvβ3/5 integrin inhibitor, was used to investigate the possible mechanism of MFG-E8. 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. 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 αVβ3/5.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the β5 integrin

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvβ5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin β5 complexes with the insulin receptor beta (IRβ) in skeletal muscle, resulting in dephosphorylation of IRβ and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between β5 and IRβ impacts IRβ phosphorylation status. We show in invitro and invivo in skeletal muscle in mice that antibody-mediated blockade of the β5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRβ resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The β5-PTP1B complex is recruited by MFGE8 to IRβ leading to termination of canonical insulin signaling. β5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and β5 in regulating insulin signaling.

An Autologous Macrophage-Based Phenotypic Transformation-Collagen Degradation System Treating Advanced Liver Fibrosis.

In advanced liver fibrosis (LF), macrophages maintain the inflammatory environment in the liver and accelerate LF deterioration by secreting proinflammatory cytokines. However, there is still no effective strategy to regulate macrophages because of the difficulty and complexity of macrophage inflammatory phenotypic modulation and the insufficient therapeutic efficacy caused by the extracellular matrix (ECM) barrier. Here, AC73 and siUSP1 dual drug-loaded lipid nanoparticle is designed to carry milk fat globule epidermal growth factor 8 (MFG-E8) (named MUA/Y) to effectively inhibit macrophage proinflammatory signals and degrade the ECM barrier. MFG-E8 is released in response to the high reactive oxygen species (ROS) environment in LF, transforming macrophages from a proinflammatory (M1) to an anti-inflammatory (M2) phenotype and inducing macrophages to phagocytose collagen. Collagen ablation increases AC73 and siUSP1 accumulation in hepatic stellate cells (HSCs) and inhibits HSCs overactivation. Interestingly, complete resolution of liver inflammation, significant collagen degradation, and HSCs deactivation are observed in methionine choline deficiency (MCD) and CCl4 models after tail vein injection of MUA/Y. Overall, this work reveals a macrophage-focused regulatory treatment strategy to eliminate LF progression at the source, providing a new perspective for the clinical treatment of advanced LF.

Synergistic effects of MFG-E8 and whey protein on mitigating d-galactose-induced sarcopenia through PI3K/AKT/PGC-1α and MAPK/ERK signaling pathways

Milk fat globule epidermal growth factor 8 (MFG-E8) and whey protein have emerged as promising bionutrient supplements for enhancing skeletal muscle mass and function. In the present study, aging-related sarcopenia rat model was employed to elucidate the effects of the combined administration of MFG-E8 and whey protein on the catabolism and anabolism of gastrocnemius protein. Combined intervention led to notable enhancements in the antioxidative stress status and mitochondrial biogenesis capacity of gastrocnemius muscle fibers in the aging rats, concomitant with a significant inhibition of lipid accumulation. Moreover, the synergistic effect of MFG-E8 and whey protein was found to exert modulatory effects on key signaling pathways, including PI3K/Akt/PGC-1α pathway and MAPK/ERK signaling pathways in the gastrocnemius muscle of the aging rats. Specifically, this combined intervention was observed to promote mitochondrial biogenesis and regulate the expression of protein anabolism and catabolism-related regulators, thereby facilitating the alleviation of mitochondrial oxidative stress and enhancing biogenesis in gastrocnemius tissues. The findings of our study provide compelling evidence for the potential of MFG-E8 as a promising dietary supplement with antisarcopenic properties to ameliorate muscle protein metabolism disorders and mitigate mitochondrial-mediated myoblast apoptosis induced by oxidative stress.

Bioactive peptides in preterm human milk: Impact of maternal characteristics and their association to neonatal outcomes.

Human milk adipokines in term babies seem partially determined by maternal factors and affect infant's development. We aimed to describe bioactive peptide concentration in very preterm human milk and associations to maternal characteristics and postnatal growth. Mothers delivering ≤32 weeks of gestation and their infant/s were recruited. At 4 weeks of lactation, an aliquot of 24-h-pooled milk was collected for exclusively breastfeeding dyads. Insulin, leptin, adiponectin, and milk fat globule epidermal growth factor-8 (MFG-E8) were measured by enzyme-linked immunoabsorbent assay in skimmed milk. One hundred mothers (28.8 ± 2.3 weeks at delivery) provided a milk sample. Milk insulin was related to gestational age, pre-pregnancy body mass index (BMI), and galactagogue treatment (final model: adjusted R2 : 0.330, p < 0.0001; adjusted β coefficients: galactagogue treatment: 0.348, p 0.001; pre-pregnancy BMI: 0.274, p 0.009; gestational age: -0.290, p 0.007). Adiponectin was higher in mothers with gestational diabetes (30.7 ± 6.5 vs. 24.8 ± 8 ng/mL, p 0.044). Leptin was associated with pre-pregnancy BMI (Spearman's ρ: 0.648, p < 0.0001) and MFG-E8 to presence of labor and multiple pregnancy (final linear regression model, R2 : 0.073, p 0.028, adjusted β coefficients: presence of labor -0.229, p 0.050; twins: -0.192, p 0.099). Milk adiponectin was associated with a greater decrease in length z-scores from birth to 28 days (Pearson's r: -0.225, p 0.032) and to discharge (Pearson's r: -0.290, p 0.003). Milk MFG-E8 was lower in milk of mothers whose babies experienced late-onset sepsis (13.3 ± 5.8 vs. 16.8 ± 6.3 μg/mL, p 0.023). Adipokines levels in preterm human milk are partially related to maternal metabolic status. Milk peptide concentration associates with early neonatal growth trajectories.

Milk fat globule epidermal growth factor 8a regulates neurogenesis in telencephalon and affects larval behavior in zebrafish.

Mfge8, a secreted glycoprotein, is a key molecule that mediates the phagocytosis of apoptotic cells. Previous research reported that Mfge8 is critical for the proliferation and differentiation of radial glia cells (RGCs) in the dentate gyrus of adult mice. The treatment of Mfge8 is also beneficial for the repair of central nervous system (CNS) injury after cerebral ischemia. This study aimed to investigate whether the expression of mfge8a in zebrafish embryos was associated with the development of CNS and larval behavior. We found that zebrafish mfge8a was initially expressed at 48 hpf, and its expression was gradually increased in the ventricular zone. Knocking down mfge8a with antisense morpholino oligonucleotides impaired both spontaneous and photo-induced swimming locomotion in the behavioral tests. The neurogenesis analysis in telencephalon showed that mfge8a morphants excessively promoted neural differentiation over self-renewal after RGCs division, and consequently depleted proliferative RGCs population during early neurogenesis. Furthermore, downregulation of mfge8a was shown to alter the expression patterns of genes associated with Notch signaling pathway. Our results demonstrated that mfge8a is involved in the maintenance of the progenitor identity of RGCs in embryonic zebrafish brain via regulating Notch signaling pathway, thereby contributing to consistent neurogenesis and locomotor development.

Milk Fat Globule Epidermal Growth Factor VIII Fragment Medin in Age-Associated Arterial Adverse Remodeling and Arterial Disease.

Medin, a small 50-amino acid peptide, is an internal cleaved product from the second discoidin domain of milk fat globule epidermal growth factor VIII (MFG-E8) protein. Medin has been reported as the most common amylogenic protein in the upper part of the arterial system, including aortic, temporal, and cerebral arterial walls in the elderly. Medin has a high affinity to elastic fibers and is closely associated with arterial degenerative inflammation, elastic fiber fragmentation, calcification, and amyloidosis. In vitro, treating with the medin peptide promotes the inflammatory phenotypic shift of both endothelial cells and vascular smooth muscle cells. In vitro, ex vivo, and in vivo studies demonstrate that medin enhances the abundance of reactive oxygen species and reactive nitrogen species produced by both endothelial cells and vascular smooth muscle cells and promotes vascular endothelial dysfunction and arterial stiffening. Immunostaining and immunoblotting analyses of human samples indicate that the levels of medin are increased in the pathogenesis of aortic aneurysm/dissection, temporal arteritis, and cerebrovascular dementia. Thus, medin peptide could be targeted as a biomarker diagnostic tool or as a potential molecular approach to curbing the arterial degenerative inflammatory remodeling that accompanies aging and disease.

MFG-E8 stabilized by deubiquitinase USP14 suppresses cigarette smoke-induced ferroptosis in bronchial epithelial cells

Milk fat globule epidermal growth factor 8 (MFG-E8) participates in a range of cellular processes, including reducing apoptosis and oxidative stress. However, its protective activity against cigarette smoke-induced ferroptosis in the pathogenesis of the chronic obstructive pulmonary disease (COPD) and the modulation of MFG-E8 remain unclear. Here, we showed that cigarette smoke diminished MFG-E8 protein levels but had no significant effect on its mRNA levels in lung tissues of humans and mice and in two human bronchial epithelial cell lines. MFG-E8 could attenuate ferroptosis induced by cigarette smoke extract (CSE) in vivo and in vitro. We identified ubiquitin-specific protease 14 (USP14) as a deubiquitinase of MFG-E8 in human bronchial epithelial cells. USP14 interacted with, deubiquitinated and stabilized MFG-E8. Furthermore, USP14 inhibited CSE-induced MFG-E8 proteasomal degradation. USP14 expression downregulated by CSE decreased MFG-E8 abundance and further reduced the antiferroptotic effect of MFG-E8. These findings suggest that USP14 is an essential regulator of MFG-E8 through the proteasomal pathway and that the USP14/MFG-E8 axis plays a critical role in regulating CSE-induced ferroptosis of bronchial epithelial cells.

MFG-E8 has guiding significance for the prognosis and treatment of sepsis

Sepsis remains a significant clinical challenge. Ferroptosis is involved in the pathogenesis of sepsis. Ferroptosis is associated with oxidative stress, and excessive oxidative stress is suppressed by milk fat globule epidermal growth factor 8 (MFG-E8) under various conditions. However, the role of MFG-E8 in sepsis-induced ferroptosis and oxidative stress is still unclear. First, we collected blood samples from patients with sepsis and detected the expression of serum MFG-E8. Then, the relationship between serum concentrations of MFG-E8 and disease severity was detected. Finally, the effects of MFG-E8 treatment on ferroptosis and oxidative stress in the livers of septic mice were determined. The expression of serum MFG-E8 in healthy subjects was notably higher than that in septic patients. In addition, when nonsurvivors and survivors of sepsis were compared, MFG-E8 levels were considerably lower in the former. The ROC curve for MFG-E8 was also generated. The area under the curve for MFG-E8 was 0.768 (95% confidence interval [CI] 0.627–0.909, p = 0.003). The patients were separated into two groups based on the MFG-E8 cut-off value of 3.86 ng/mL. According to the Kaplan‒Meier survival analysis, patients with low MFG-E8 levels had a significantly decreased 28-day survival rate compared with patients with high MFG-E8 levels. High MFG-E8 levels were substantially related to a decreased risk of death, as demonstrated by the Cox proportional hazard model that we utilized. In addition, compared with sham mice, septic mice exhibited liver and kidney damage, and MFG-E8 may have protective effects. The survival study indicated that MFG-E8 could effectively improve the survival rate of septic mice. Treatment with MFG-E8 suppresses oxidative stress and ferroptosis in the livers of septic mice. Serum MFG-E8 levels are lower in septic patients and are negatively related to disease severity. Treatment with MFG-E8 suppresses oxidative stress and ferroptosis in the livers of septic mice, contributing to significantly improved survival in septic mice. These findings showed that MFG-E8 could be a new sepsis predictive biomarker. MFG-E8 may have therapeutic potential in the treatment of sepsis.

Elevated MFG-E8 in CSF in the Early Stage Indicates Rapid Recovery of Mild Aneurysmal SAH Patients.

Background Aneurysmal subarachnoid hemorrhage (aSAH) can impair blood perfusion in brain tissue and cause adverse effects. Microglia, which are the inherent immune cells of the brain, significantly activate and play a role in phagocytosis, anti-inflammatory, proinflammatory, and damage repair in this process. Milk fat globule epidermal growth factor 8 (MFG-E8) is the bridging molecule of this process and mediates the activation and biological effects of microglia. Methods We obtained cerebrospinal fluid (CSF) from patients with aSAH at various times (the third day, seventh day, and ninth day) as well as from patients in the control cohort. MFG-E8 protein levels in CSF were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we evaluated the GCS and GOS of aSAH patients on admission and on the third day, seventh day, ninth day, and at discharge. Then, we analyzed the association between the levels of MFG-E8 and the changes in GCS and GOS. Results MFG-E8 expression rose in the early stage on the third day and reached equilibrium around day 7 and day 9. The levels of MFG-E8 on the third day were associated with the change in GOS on the seventh day (r = 0.644, p = 0.018) and ninth day (r = 0.572, p = 0.041) compared with admission but were not correlated with the change on day 3 or at discharge. The levels of MFG-E8 were not correlated with any change in GCS. Conclusions We found that aSAH resulted in an upregulation of MFG-E8 in CSF. Moreover, high MFG-E8 levels in the early stage indicated a rapid recovery of mild aSAH patients.

Preventing SARS-CoV-2 Infection Using Anti-spike Nanobody-IFN-β Conjugated Exosomes.

PurposeTo inhibit the transmission of SARS-CoV-2, we developed engineered exosomes that were conjugated with anti-spike nanobodies and type I interferon β (IFN-β). We evaluated the efficacy and potency of nanobody-IFN-β conjugated exosomes to treatment of SARS-CoV-2 infection.MethodsMilk fat globule epidermal growth factor 8 (MFG-E8) is a glycoprotein that binds to phosphatidylserine (PS) exposed on the exosomes. We generated nanobody-IFN-β conjugated exosomes by fusing an anti-spike nanobody and IFN-β with MFG-E8. We used the SARS-CoV-2 pseudovirus with the spike of the D614G mutant that encodes ZsGreen to mimic the infection process of the SARS-CoV-2. The SARS-CoV-2 pseudovirus was infected with angiotensin-converting enzyme-2 (ACE2) expressing adenocarcinomic human alveolar basal epithelial cells (A549) or ACE2 expressing HEK-blue IFNα/β cells in the presence of nanobody-IFN-β conjugated exosomes. By assessing the expression of ZsGreen in target cells and the upregulation of interferon-stimulated genes (ISGs) in infected cells, we evaluated the anti-viral effects of nanobody-IFN-β conjugated exosomes.ResultsWe confirmed the anti-spike nanobody and IFN-β expressions on the exosomes. Exosomes conjugated with nanobody-hIFN-β inhibited the interaction between the spike protein and ACE2, thereby inhibiting the infection of host cells with SARS-CoV-2 pseudovirus. At the same time, IFN-β was selectively delivered to SARS-CoV-2 infected cells, resulting in the upregulation of ISGs expression.ConclusionExosomes conjugated with nanobody-IFN-β may provide potential benefits in the treatment of COVID-19 because of the cooperative anti-viral effects of the anti-spike nanobody and the IFN-β.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11095-022-03400-0.

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

BackgroundAge‐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 ResultsTo 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.ConclusionsThus, 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.