Milk Fat globule-EGF Factor Research Articles

Rapid increase of MFGE8 secretion from endometrial epithelial cells is an indicator of extracellular vesicle mediated embryo maternal dialogue

Successful embryo implantation relies on synchronized dialog between the embryo and endometrium, and the role of extracellular vesicles (EVs) in facilitating this cross-talk has been recently established. In our previous study, milk fat globule-EGF factor 8 protein (MFGE8) was identified as increasing in receptive endometrial epithelial cells (EECs) in response to trophoblastic EVs. However, the dynamics of MFGE8 protein in this context are not completely understood. Therefore, we examined its expression and secretion in EECs exposed to estrogen, progesterone, and trophoblastic EVs to gain deeper insights into its potential as an indicator of EV-mediated embryo-maternal dialogue. Our findings revealed that MFGE8 secretion is sensitive to estrogen and progesterone, and that trophoblastic EVs stimulate their release in both receptive and non-receptive EECs. Furthermore, trophoblast EV function was dose and time-dependent. Notably, the secretion of MFGE8 increased within a short timeframe of 30 min after addition of EVs, suggesting the possibility of rapid processes such as binding, fusion or internalization of trophoblastic EVs within EECs. Interestingly, MFGE8 released from EECs was associated with EVs, suggesting increased EV secretion from EECs in response to embryonic signals. In conclusion, increased MFGE8 secretion in this embryo implantation model can serve as an indicator of EV-mediated embryo-maternal dialogue.

Astrocyte-derived MFG-E8 facilitates microglial synapse elimination in Alzheimer's disease mouse models.

Region-specific synapse loss is an early pathological hallmark in Alzheimer's disease (AD). Emerging data in mice and humans highlight microglia, the brain-resident macrophages, as cellular mediators of synapse loss; however, the upstream modulators of microglia-synapse engulfment remain elusive. Here, we report a distinct subset of astrocytes, which are glial cells essential for maintaining synapse homeostasis, appearing in a region-specific manner with age and amyloidosis at onset of synapse loss. These astrocytes are distinguished by their peri-synaptic processes which are 'bulbous' in morphology, contain accumulated p62-immunoreactive bodies, and have reduced territorial domains, resulting in a decrease of astrocyte-synapse coverage. Using integrated in vitro and in vivo approaches, we show that astrocytes upregulate and secrete phagocytic modulator, milk fat globule-EGF factor 8 (MFG-E8), which is sufficient and necessary for promoting microglia-synapse engulfment in their local milieu. Finally, we show that knocking down Mfge8 specifically from astrocytes using a viral CRISPR-saCas9 system prevents microglia-synapse engulfment and ameliorates synapse loss in two independent amyloidosis mouse models of AD. Altogether, our findings highlight astrocyte-microglia crosstalk in determining synapse fate in amyloid models and nominate astrocytic MFGE8 as a potential target to ameliorate synapse loss during the earliest stages of AD.

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.

COMT Variants are Associated With Breast and Nipple Pain

Estimates suggest that only 24.9% of infants born in 2019 were exclusively breastfed before 6 months of age, despite the known health benefits of exclusive breastfeeding. Breast and nipple pain is one of the primary determinants of exclusive breastfeeding. Environmental contributions to breastfeeding success have been reported extensively in the literature, but the contribution(s) of maternal genetics has yet to be discovered. The purpose of the study was to identify an association between pain and lactation-related gene variants with exclusive breastfeeding determinants. We selected 4 genes having single nucleotide polymorphisms (SNPs) with potential functional significance in breastfeeding and pain: prolactin receptor (PRLR), oxytocin receptor (OXTR), catechol-O-methyltransferase (COMT), and milk fat globule epidermal growth factor and factor V/VIII domain containing (MFGE8). We performed a cross-sectional secondary analysis of a longitudinal randomized controlled trial study, Promoting Self-Management of Breast and Nipple Pain with Biomarkers and Technology for Breastfeeding Women (NCT05262920). Breast and nipple pain, perceived insufficient milk, and breastfeeding self-efficacy were examined using total scale scores for the Brief Pain Inventory, Visual Analog Scale, H&H Lactation Scale, and the Breastfeeding Self-efficacy Scale-short form, respectively. Of the candidate genes examined, SNPs within COMT were significantly associated with breastfeeding-related outcomes. Specifically, COMT rs4633 and rs4680 minor allele carriers (T, A) reported higher breast and nipple pain intensity than women homozygous for the major allele (C, G). COMT is the most widely researched “pain gene” and has been linked to cold, postoperative, and postpartum pain. This study is the first to identify a contribution of COMT variants to breast and nipple pain and, as a result, to breastfeeding exclusivity. PerspectiveTwo SNPs in the pain gene COMT are associated with breast and nipple pain. Clinically, a minor allele in COMT rs4633 and rs4680 may increase a woman’s rating of moderate breast and nipple pain. Trial registrationPROMPT was registered in ClinicalTrials.gov (protocol #NCT05262920).

Extracellular vesicle-derived silk fibroin nanoparticles loaded with MFGE8 accelerate skin ulcer healing by targeting the vascular endothelial cells

BackgroundReduced supplies of oxygen and nutrients caused by vascular injury lead to difficult-to-heal pressure ulcers (PU) in clinical practice. Rapid vascular repair in the skin wound is the key to the resolution of this challenge, but clinical measures are still limited. We described the beneficial effects of extracellular vesicle-derived silk fibroin nanoparticles (NPs) loaded with milk fat globule EGF factor 8 (MFGE8) on accelerating skin blood vessel and PU healing by targeting CD13 in the vascular endothelial cells (VECs).MethodsCD13, the specific targeting protein of NGR, and MFGE8, an inhibitor of ferroptosis, were detected in VECs and PU tissues. Then, NPs were synthesized via silk fibroin, and MFGE8-coated NPs (NPs@MFGE8) were assembled via loading purified protein MFGE8 produced by Chinese hamster ovary cells. Lentivirus was used to over-express MFGE8 in VECs and obtained MFGE8-engineered extracellular vesicles (EVs-MFGE8) secreted by these VECs. The inhibitory effect of EVs-MFGE8 or NPs@MFGE8 on ferroptosis was detected in vitro. The NGR peptide cross-linked with NPs@MFGE8 was assembled into NGR-NPs@MFGE8. Collagen and silk fibroin were used to synthesize the silk fibroin/collagen hydrogel. After being loaded with NGR-NPs@MFGE8, silk fibroin/collagen hydrogel sustained-release carrier was synthesized to investigate the repair effect on PU in vivo.ResultsMFGE8 was decreased, and CD13 was increased in PU tissues. Similar to the effect of EVs-MFGE8 on inhibiting ferroptosis, NPs@MFGE8 could inhibit the mitochondrial autophagy-induced ferroptosis of VECs. Compared with the hydrogels loaded with NPs or NPs@MFGE8, the hydrogels loaded with NGR-NPs@MFGE8 consistently released NGR-NPs@MFGE8 targeting CD13 in VECs, thereby inhibiting mitochondrial autophagy and ferroptosis caused by hypoxia and accelerating wound healing effectively in rats.ConclusionsThe silk fibroin/collagen hydrogel sustained-release carrier loaded with NGR-NPs@MFGE8 was of great significance to use as a wound dressing to inhibit the ferroptosis of VECs by targeting CD13 in PU tissues, preventing PU formation and promoting wound healing.Graphical

Liver Wound Healing Is Associated with An Increase in Mfge8 Expression in Hepatocytes

Being recognized as a typical pathological feature of liver diseases, apoptosis-induced hepatocyte injury leading to sterile inflammation drives a wound-healing path in vivo. We and other researchers reported that Milk fat globule-EGF factor 8 (MFGE8) preserves tissue homeostasis, protects against tissue injury and inflammation, and attenuates nonalcoholic fatty liver disease and fibrosis. However, it is unclear whether and how MFGE8 is involved in liver regeneration from hepatocyte-apoptosis-induced injury. Our aim is to study current knowledge to this extent, we created a novel triple-transgenic mouse model, an inducible hepatocyte-specific apoptosis phenotype (namely, 3XTg-iHAP), in which the inducible hepatocyte-specific FasL expression is under control of the combined LoxP-Albumin-Cre system and rtTA Tet-On regulatory element. The liver tissues from 3XTg-iHAP and their littermates were collected at 8 h, 24 h, 48 h, 72 h, and 5 D after doxycycline (Dox, 5mg/kg, s.c., single dose) treatment and subjected to histological examination, RNAScope in situ hybridization (ISH) and qRT-PCR analysis. We first confirmed that the 3XTg-iHAP mice developed normal, viable, and fertile. Upon Dox treatment, qRT-PCR confirmed FasL expression was specifically induced in the liver tissues of 3XTg-iHAP mice (male, 7 weeks) but not in their littermates. H&E sections show the induction of hepatocyte apoptosis as early as 24 h followed by apoptosis-induced sterile inflammation and injury, hepatocyte mitosis, and regeneration in the liver parenchyma of 3XTg-iHAP mice. In the ISH assay, we found that (1) In littermate control mice, Mfge8 transcripts were localized in the intrahepatic bile duct epithelial cells (i.e., cholangiocytes) within the portal area. (2) In Dox-treated 3XTg-iHAP mice, we observed a profound increase of Mfge8 gene expression in parenchymal hepatocytes of the liver featured with apoptosis-associated sterile injury. In summary, we validated a novel 3XTg-iHAP mouse liver wound-healing model. The Mfge8 gene is constitutively expressed in the bile duct epithelial cells under the normal physiological condition, while upregulated in hepatocytes in response to the apoptosis-induced injury and repair. Our results suggest that liver wound healing is associated with an increase in Mfge8 expression in hepatocytes. The underlying mechanisms of whether and how MFG-E8 promotes the liver wound healing process need to be further studied. Grants are supported by NIH and the U.S. Department of Veterans Affairs. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting antigens to phosphatidylserine triggers rapid T- and B-cell responses

Abstract Phosphatidylserine (PS) is a phospholipid that is present in each cell membrane. In intact cells, PS is kept intracellularly, in dying cells, extracellular vesicles (EVs) and activated platelets, PS flips outside where it can be accessed by PS-binding reagents, such as Milk fat globule-EGF factor 8 (MFG-E8). In previous studies, we have established in vivouse of fluorescent MFG-E8 to label and characterize EVs. Here, we measured the antigen-specific immune responses elicited by targeting antigen (Ag) to PS +structures, presumably EVs via MFG-E8 as a novel vaccination strategy. In contrast to non-targeted conventional Ag, PS-targeting by fusing Ag to MFG-E8 caused accumulation in the marginal zone of lymphoid follicles soon after injection. Hours later, Ag-MFG-E8 was deposited on the follicular dendritic cell network without the need of previous primary immunization. Mice immunized with PS-targeted Ag/Alum induced long-lasting germinal centers (GCs) and significantly more GC B cells. Furthermore, it resulted in an approx. 13,000-fold increase in Ag-specific IgG-levels and strongly accelerated antibody class switch recombination as compared to immunization with conventional Ag/Alum. Ag targeted to PS also induced more efficient CD8 T cell activation and IFN-gamma production post immunization. Therefore, targeting Ag to EVs by harnessing the PS-binding properties of MFG-E8 might turn out to be of advantage for the induction of very rapid and long-lasting immune responses. This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research foundation) – Project-ID 210592381 - SFB 1054 B03 to Thomas Brocker and Z02 to Jan Kranich.

Acute high-fat diet impairs macrophage-supported intestinal damage resolution.

Chronic exposure to high-fat diets (HFD) worsens intestinal disease pathology, but acute effects of HFD in tissue damage remain unclear. Here, we used short-term HFD feeding in a model of intestinal injury and found sustained damage with increased cecal dead neutrophil accumulation, along with dietary lipid accumulation. Neutrophil depletion rescued enhanced pathology. Macrophages from HFD-treated mice showed reduced capacity to engulf dead neutrophils. Macrophage clearance of dead neutrophils activates critical barrier repair and antiinflammatory pathways, including IL-10, which was lost after acute HFD feeding and intestinal injury. IL-10 overexpression restored intestinal repair after HFD feeding and intestinal injury. Macrophage exposure to lipids from the HFD prevented tethering and uptake of apoptotic cells and Il10 induction. Milk fat globule-EGF factor 8 (MFGE8) is a bridging molecule that facilitates macrophage uptake of dead cells. MFGE8 also facilitates lipid uptake, and we demonstrate that dietary lipids interfere with MFGE8-mediated macrophage apoptotic neutrophil uptake and subsequent Il10 production. Our findings demonstrate that HFD promotes intestinal pathology by interfering with macrophage clearance of dead neutrophils, leading to unresolved tissue damage.

Identification and antioxidative mechanism of novel mitochondria-targeted MFG-E8 polypeptides in virtual screening and in vitro study

Milk fat globule-EGF factor VIII (MFG-E8) has been identified as an important source of bioactive peptides, which may exert a pivotal role in regulating biologic redox equilibrium. However, the composition of MFG-E8 polypeptides and their mechanisms on mitigating sarcopenia remain unknown. The aim of this study was to identify the composition of MFG-E8 polypeptides and its effects against oxidative stress in dexamethasone-induced L6 cell injury. Simulated digestion in vitro and liquid chromatography-tandem mass spectrometry were used in this investigation. A total of 95 peptides were identified during complete simulated digestion; among them, the contents of 21 peptides were analyzed, having been determined to exceed 1%. Molecular docking assay found that IDLG, KDPG, YYR, and YYK exhibited high binding affinity with keap1. MTT, dichlorodihydrofluorescein diacetate, mito- and lyso-tracker, and transmission electron microscope assay demonstrated that IDLG and KDPG can alleviate oxidative stress-injured L6 cell vitality, mitochondria activity, vacuolation, and function decrease, and increased autophagy, thereby improving mitochondrial homeostasis. From a molecular perspective, IDLG and KDPG can decrease the expression of keap1 and increase the expression of Nrf2, HO-1, and PGC-1α. Therefore, MFG-E8-derived IDLG and KDPG could be potential polypeptides countering oxidative stress in the treatment of sarcopenia, via the keap1/Nrf2/HO-1 signaling pathway.

The cytoprotection of milk-derived MFG-E8 on mitochondria-injured L6 cell via mediation of Akt/bcl-2/bax-caspase-3 signaling pathway

Mitochondria-dependent myoblast apoptosis induced by oxidative stress and decrease in successful protein synthesis play a crucial role in loss of muscle mass and functionality further to result in sarcopenia. Here, we investigated the relationship between milk fat globule-EGF factor Ⅷ (MFG-E8) and mitochondrial-dependent apoptosis pathway. MFG-E8 exert cytoprotection through increasing L6 cells survival/apoptotic ratio, increasing mitochondrial membrane potential and regulating S and G2/M phases. By observing cell ultrastructure, MFG-E8 improved mitochondrial homeostasis mainly through decreasing cytochrome-c release, expression of caspase-9 and caspase-3, mitochondrial vacuolation and mitophagy thereby inhibiting cell apoptosis. From molecular perspective, MFG-E8 repaired mitochondria fragmentation by increasing mitochondrial DNA replication and regulated expression of key mitochondrial-apoptotic factors (upregulation: B-cell lymphoma-2 like 1 (bcl2l1), bcl2l2, cyclooxygenase-1 and mitochondrial uncoupling protein-2; Downregulation: p21 and p53) further to improve mitochondrial function and inhibit apoptosis. Moreover, MFG-E8 inhibited mitochondrial-dependent apoptosis via Akt/bcl-2/bax/caspase-3 signaling cascades. Taken together, our research provided a promising approach for deep exploration of MFG-E8 on cytoprotection against mitochondrial injury and mitochondrial-mediated apoptosis and application of anti-apoptosis in alleviating sarcopenia.

MicroRNA-34b-5p binds enhancer of zeste 2 to inhibit milk fat globule-EGF factor 8 expression, affecting liver fibrosis

Activated hepatic stellate cells (HSCs) are considered the major drivers in the process of hepatic fibrosis. This study intends to explore the mechanism underlying microRNA (miR)-34b-5p effects over liver fibrosis through the enhancer of zeste 2 (EZH2)/milk fat globule-EGF factor 8 (MFGE8) axis in HSCs. A liver fibrosis model was generated by carbon tetrachloride (CCl4) in C57BL/6J mice and subjected to histological examinations and detection of HSC activation and miR-34b-5p/EZH2/MFGE8 expression. Primary HSCs were treated with transforming growth factor (TGF)-β and tested for proliferation, activation, and expression of fibrosis-related factors. A dual luciferase reporter assay was performed for confirming the targeted relationship between miR-34b-5p and EZH2. Chromatin immunoprecipitation was used to measure EZH2 enrichment in the MFGE8 promoter region. We found that miR-34b-5p was lowly expressed in the CCl4-induced mouse model. Overexpression of miR-34b-5p suppressed both TGF-β-induced HSC proliferation and the expression of fibrosis-related factors and HSC activation markers. A dual luciferase assay showed a binding relationship between miR-34b-5p and EZH2. Overexpression of miR-34b-5p reduced TGF-β-induced HSC activation by inhibiting EZH2 to promote MFGE8 expression. Overexpression of miR-34b-5p inhibited liver fibrosis in vivo through the EZH2/MFGE8 axis. Conclusively, overexpressing miR-34b-5p reduced TGF-β-induced HSC activation by inhibiting EZH2 and thereby promoting MFGE8 expression, and inhibited liver fibrosis in vivo through the EZH2/MFGE8 axis.

Does the RGD region of certain proteins affect metabolic activity?

A better understanding of the role of mineralized tissues and their associated factors in governing whole-body metabolism should be of value toward informing clinical strategies to treat mineralized tissue and metabolic disorders, such as diabetes and obesity. This perspective provides evidence suggesting a role for the arginine-glycine-aspartic acid (RGD) region, a sequence identified in several proteins secreted by bone cells, as well as other cells, in modulating systemic metabolic activity. We focus on (a) two of the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family genes/proteins, bone sialoprotein (BSP) and osteopontin (OPN), (b) insulin-like growth factor-binding protein-1 & 2 (IGFBP-1, IGFBP-2) and (c) developmental endothelial locus 1 (DEL1) and milk fat globule–EGF factor-8 (MFG-E8). In addition, for our readers to appreciate the mounting evidence that a multitude of bone secreted factors affect the activity of other tissues, we provide a brief overview of other proteins, to include fibroblast growth factor 23 (FGF23), phosphatase orphan 1 (PHOSPHO1), osteocalcin (OCN/BGLAP), tissue non-specific alkaline phosphatase (TNAP) and acidic serine aspartic-rich MEPE-associated motif (ASARM), along with known/suggested functions of these factors in influencing energy metabolism.

Genome-wide identification of long noncoding RNA genes and their potential association with mammary gland development in water buffalo.

Water buffalo, an important domestic animal in tropical and subtropical regions, play an important role in agricultural economy. It is an important source for milk, meat, horns, skin, and draft power, especially its rich milk that is the great source of cream, butter, yogurt, and many cheeses. In recent years, long noncoding RNAs (lncRNAs) have been reported to play pivotal roles in many biological processes. Previous studies for the mammary gland development of water buffalo mainly focus on protein coding genes. However, lncRNAs of water buffalo remain poorly understood, and the regulation relationship between mammary gland development/milk production traits and lncRNA expression is also unclear. Here, we sequenced 22 samples of the milk somatic cells from three lactation stages and integrated the current annotation and identified 7,962 lncRNA genes. By comparing the lncRNA genes of the water buffalo in the early, peak, and late different lactation stages, we found that lncRNA gene lnc-bbug14207 displayed significantly different expression between early and late lactation stages. And lnc-bbug14207 may regulate neighboring milk fat globule-EGF factor 8 (MFG-E8) and hyaluronan and proteoglycan link protein 3 (HAPLN3) protein coding genes, which are vital for mammary gland development. This study provides the first genome-wide identification of water buffalo lncRNAs and unveils the potential lncRNAs that impact mammary gland development.

Involvement of phosphatidylserine receptors in the skeletal muscle regeneration: therapeutic implications.

Sarcopenia is a progressive loss of muscle mass and strength with a risk of adverse outcomes such as disability, poor quality of life, and death. Increasing evidence indicates that diminished ability of the muscle to activate satellite cell‐dependent regeneration is one of the factors that might contribute to its development. Skeletal muscle regeneration following myogenic cell death results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibres. Satellite cell differentiation is not a satellite cell‐autonomous process but depends on signals provided by the surrounding cells. Infiltrating macrophages play a key role in the process partly by clearing the necrotic cell debris, partly by producing cytokines and growth factors that guide myogenesis. At the beginning of the muscle regeneration process, macrophages are pro‐inflammatory, and the cytokines produced by them trigger the proliferation and differentiation of satellite cells. Following the uptake of dead cells, however, a transcriptionally regulated phenotypic change (macrophage polarization) is induced in them resulting in their transformation into healing macrophages that guide resolution of inflammation, completion of myoblast differentiation, myoblast fusion and growth, and return to homeostasis. Impaired efferocytosis results in delayed cell death clearance, delayed macrophage polarization, prolonged inflammation, and impaired muscle regeneration. Thus, proper efferocytosis by macrophages is a determining factor during muscle repair. Here we review that both efferocytosis and myogenesis are dependent on the cell surface phosphatidylserine (PS), and surprisingly, these two processes share a number of common PS receptors and signalling pathways. Based on these findings, we propose that stimulating the function of PS receptors for facilitating muscle repair following injury could be a successful approach, as it would enhance efferocytosis and myogenesis simultaneously. Because increasing evidence indicates a pathophysiological role of impaired efferocytosis in the development of chronic inflammatory conditions, as well as in impaired muscle regeneration both contributing to the development of sarcopenia, improving efferocytosis should be considered also in its management. Again applying or combining those treatments that target PS receptors would be expected to be the most effective, because they would also promote myogenesis. A potential PS receptor‐triggering candidate molecule is milk fat globule‐EGF‐factor 8 (MFG‐E8), which not only stimulates PS‐dependent efferocytosis and myoblast fusion but also promotes extracellular signal‐regulated kinase (ERK) and Akt activation‐mediated cell proliferation and cell cycle progression in myoblasts.

Abstract 373: Senescence Drives Changes In Vesicle Secretion And Extracellular Matrix Organisation To Enhance Vascular Amyloidosis

Background: Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid fragment of milk fat globule EGF-factor 8 (MFGE8). Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid metabolism and accumulation in the extracellular matrix (ECM). MFGE8 has been identified as EV cargo in several cell types, including vascular smooth muscle cells (VSMCs). To determine the mechanisms of AMA formation with age, we explored the impact of VSMC senescence, EV secretion and ECM remodelling on medin accumulation. Methods: Primary human VSMCs were used for in vitro experiments, including EV isolation and synthesis of decellularised ECM for Western blotting, immunofluorescence and mass spectrometry analysis. Results: Medin was detected in EVs secreted from VSMCs. Small, round medin aggregates colocalised with EV markers in the ECM and medin was shown on the surface of EVs deposited in the ECM. Decreasing VSMC EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age. VSMC senescence increased EV secretion, increased EV medin loading and triggered extracellular deposition of medin in fibrillar form. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition. These changes led to enhanced EV-ECM binding in the senescent ECM and accelerated aggregation of medin into amyloid fibrils. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalised with EVs and medin. Knock-down of HSPG2 decreased medin fibril formation in the senescent ECM. Conclusions: Medin is secreted into the ECM by VSMC-derived EVs, which enhance its aggregation. Senescence induces changes in EV secretion and cargo and ECM composition. Together, these changes trigger accumulation of fibrillar medin, contributing to the age-associated development of AMA.

MFG-E8 alleviates intervertebral disc degeneration by suppressing pyroptosis and extracellular matrix degradation in nucleus pulposus cells via Nrf2/TXNIP/NLRP3 axis

Intervertebral disc degeneration (IVDD) is a chronic age-related degenerative disease accompanied by complex pathophysiological mechanisms. Increasing evidence indicates that NLRP3 inflammasome mediated pyroptosis of nucleus pulposus (NP) cells displays an important role in the pathological progression of IVDD. Milk fat globule-EGF factor-8 (MFG-E8) is an endogenously secreted glycoprotein with beneficial effects of anti-inflammatory, antioxidant, and modulation of NLRP3 inflammasome. However, the effect of MFG-E8 on IVDD remains unclear. In this study, our purpose is to clarify the expression changes of MFG-E8 in the IVDD process and explore the role and mechanism of MFG-E8. We found that MFG-E8’s expression was reduced in degraded nucleus pulposus tissues of humans and rats as well as hydrogen peroxide (H2O2)-treated NP cells. Exogenous supplementation of MFG-E8 could rescue H2O2-induced oxidative stress, mitochondrial dysfunction, and NLRP3 inflammasome activation and protect NP cells from pyroptosis and extracellular matrix (ECM) degradation. Mechanistically, Nrf2/TXNIP/NLRP3 axis plays a crucial role in MFG-E8-mediated suppression of the above-pathological events. In vivo, we established a rat intervertebral disc acupuncture model and found that MFG-E8 administration effectively alleviated IVDD development by imageological and histomorphological evaluation. Overall, our findings revealed the internal mechanisms underlying MFG-E8 regulation in NP cells and its intrinsic value for IVDD therapy.

Synaptic pruning of murine adult-born neurons by microglia depends on phosphatidylserine.

New neurons, continuously added in the adult olfactory bulb (OB) and hippocampus, are involved in information processing in neural circuits. Here, we show that synaptic pruning of adult-born neurons by microglia depends on phosphatidylserine (PS), whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and the functional maturation of adult-born neurons. The MFG-E8D89E-based genetic approach shown in this study has broad applications for understanding the biology of PS-mediated phagocytosis in vivo.

MFG-E8 Maintains Cellular Homeostasis by Suppressing Endoplasmic Reticulum Stress in Pancreatic Exocrine Acinar Cells.

Excessive endoplasmic reticulum (ER) stress contributes significantly to the pathogenesis of exocrine acinar damage in acute pancreatitis. Our previous study found that milk fat globule EGF factor 8 (MFG-E8), a lipophilic glycoprotein, alleviates acinar cell damage during AP via binding to αvβ3/5 integrins. Ligand-dependent integrin-FAK activation of STAT3 was reported to be of great importance for maintaining cellular homeostasis. However, MFG-E8’s role in ER stress in pancreatic exocrine acinar cells has not been evaluated. To study this, thapsigargin, brefeldin A, tunicamycin and cerulein + LPS were used to induce ER stress in rat pancreatic acinar cells in vitro. L-arginine- and cerulein + LPS-induced acute pancreatitis in mice were used to study ER stress in vivo. The results showed that MFG-E8 dose-dependently inhibited ER stress under both in vitro and in vivo conditions. MFG-E8 knockout mice suffered more severe ER stress and greater inflammatory response after L-arginine administration. Mechanistically, MFG-E8 increased phosphorylation of FAK and STAT3 in cerulein + LPS-treated pancreatic acinar cells. The presence of specific inhibitors of αvβ3/5 integrin, FAK or STAT3 abolished MFG-E8’s effect on cerulein + LPS-induced ER stress in pancreatic acinar cells. In conclusion, MFG-E8 maintains cellular homeostasis by alleviating ER stress in pancreatic exocrine acinar cells.

Potential influences of expression levels of MFGE8 and HMGB1 on the intestinal mucosal barrier function and inflammatory response after blunt abdominal injury in rats.

ABSTRACTPurpose:To explore the influence of milk fat globule-EGF factor 8 protein (MFGE8) on blunt abdominal injury in Sprague Dawley (SD) rats through the RhoA/ROCK signaling pathway.Methods:The blunt abdominal injury model was generated in SD rats. A total of 44 rats was randomly assigned into three groups. Rat blunt abdominal injury was assessed by the abbreviated injury scale (AIS). The rats were sacrificed for observing the morphology of the abdominal cavity and intestines. Hematoxylin and eosin staining was performed to visualize the pathological changes of rat intestines. Positive expressions of MFGE8 and high mobility group box 1 (HMGB1) in rat intestines were examined by immunohistochemical staining. Protein levels were determined by Western blot. Serum levels of tumor necrosis factor α (TNF-α), IL-1β, IL-6 and malondialdehyde (MDA) were measured by enzyme linked immunosorbent assay (ELISA).Results:Blunt abdominal injury resulted in inflammatory response of intestinal tissues, increased serum levels of TNF-α, IL-1β, IL-6 and MDA, upregulation of HMGB1, RhoA and ROCK2, and downregulation of MFGE8 in rats, which were significantly alleviated by intervention of rhMFGE8.Conclusions:MFGE8 protects the intestinal mucosal barrier function after blunt abdominal injury in rats by downregulating HMGB1. Moreover, it alleviates inflammatory response and oxidative stress caused by blunt abdominal injury in rats through downregulating RhoA and ROCK.

Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model.

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-β) expression in lungs, together with an increased expression of TGF-β related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin β3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.