MFG-E8 Regulates Angiogenesis in Cutaneous Wound Healing

Our research group recently demonstrated that pericytes are major sources of the secreted glycoprotein and integrin ligand lactadherin (MFG-E8) in B16 melanoma tumors, and that MFG-E8 promotes angiogenesis via enhanced PDGF–PDGFRβ signaling mediated by integrin–growth factor receptor crosstalk. However, sources of MFG-E8 and its possible roles in skin physiology are not well characterized. The objective of this study was to characterize the involvement of MFG-E8 in skin wound healing. In the dermis of normal murine and human skin, accumulations of MFG-E8 were found around CD31⁺ blood vessels, and MFG-E8 colocalized with PDGFRβ⁺, αSMA⁺, and NG2⁺ pericytes. MFG-E8 protein and mRNA levels were elevated in the dermis during full-thickness wound healing in mice. MFG-E8 was diffusely present in granulation tissue and was localized around blood vessels. Wound healing was delayed in MFG-E8 knockout mice, compared with the wild type, and myofibroblast and vessel numbers in wound areas were significantly reduced in knockout mice. Inhibition of MFG-E8 production with siRNA attenuated the formation of capillary-like structures in vitro. Expression of MFG-E8 in fibrous human granulation tissue with scant blood vessels was less than that in granulation tissue with many blood vessels. These findings suggest that MFG-E8 promotes cutaneous wound healing by enhancing angiogenesis.Wound healing is a dynamic process involving angiogenesis, production of soluble mediators and extracellular matrix, and migration of various types of cells, including keratinocytes, fibroblasts, macrophages, and leukocytes. Dysregulation of this interactive process may result in delayed wound healing, as is seen in chronic skin ulcers or scarring. Wound healing has three temporally overlapping phases: inflammation, tissue formation, and remodeling.^1,2 The inflammation phase occurs immediately after wounding. It is characterized by hypoxia with fibrin clot formation, as well as recruitment of neutrophils and platelets. Tissue formation occurs 2 to 10 days later and is characterized by epithelialization, formation of granulation tissue and new blood vessels, and accumulation of macrophages and fibroblasts. Activated macrophages release growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and initiate angiogenesis. PDGF receptor β (PDGFRβ) signaling is essential for angiogenesis and for recruitment, proliferation, and normal function of fibroblasts and pericytes during the tissue-formation phase.³ Blockade of VEGF receptor signaling and PDGFRβ signaling inhibits angiogenesis and results in delayed wound healing,^3,4 indicating that angiogenesis is critical for normal wound healing.The secreted glycoprotein lactadherin was initially identified as a component of milk fat globules and is here referred to as milk fat globule-EGF factor 8 (MFG-E8); other names in the literature include secreted protein containing epidermal growth factor (EGF) repeats and discoidin/F5/8 domains 1, or SED1. MFG-E8 comprises two N-terminal EGF-like domains, and two C-terminal discoidin-like domains (C1 and C2) share homology with blood coagulation factors V and VIII.^5–9 One EGF-like domain (E2) contains an RGD consensus integrin-binding motif, and MFG-E8 binds to integrin αvβ3/5.^7–11 The C-terminal domains of MFG-E8 can bind to negatively charged and oxidized phospholipids,^12,13 facilitating opsonization of apoptotic cells for uptake by phagocytes.^10,14 This process has been reported to contribute to autoimmunity, mastitis, sepsis, atherosclerosis, and Alzheimer disease.^15–19 Interactions of MFG-E8 with CD51 (integrin αv) have also been implicated in regulation of angiogenesis and mammary gland branching,^11,20 and interactions mediated via the C1 domain are thought to be important for sperm–egg binding and collagen turnover.^21,22Our research group has previously demonstrated that MFG-E8 enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice.²³ We determined that pericytes and/or pericyte precursors are important sources of MFG-E8 in vivo, that MFG-E8 enhances angiogenesis via actions on pericytes as well as endothelial cells (ECs), and that MFG-E8 can be effectively targeted with therapeutic benefit.²³ In murine melanomas and in retinas of mice with oxygen-induced retinopathy, MFG-E8 colocalized with pericytes rather than with ECs, and pericytes purified from tumors contained large amounts of MFG-E8 mRNA. Tumor- and retinopathy-associated angiogenesis was diminished in MFG-E8 knockout (KO) mice, and pericyte coverage of neovessels was also reduced. Inhibition of MFG-E8 production by pericyte/pericyte precursor-like 10T1/2 cells using siRNAs, or inhibition of MFG-E8 action with some anti–MFG-E8 antibodies, attenuated PDGF-BB–induced 10T1/2 cell migration, but did not affect proliferation or differentiation.²³ We have also determined that MFG-E8 produced by 10T1/2 cells associated with integrin αv and PDGFRβ on cell surfaces after PDGF-BB treatment, altered the distribution of PDGFRβ within cells and delayed PDGF-BB–stimulated degradation of PDGFRβ, thereby enhancing PDGFRβ signaling mediated by integrin–growth factor receptor crosstalk.²⁴In a study of MFG-E8, epithelial tissues, and wound healing, Bu et al²⁵ found that MFG-E8 promotes the migration of intestinal epithelial cells via a PKCε-dependent mechanism engaged by binding of MFG-E8 to phosphatidylserine. Their findings indicate that MFG-E8 is involved in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing. The possible role of MFG-E8 in cutaneous wound healing has not been studied previously. In the present study, we analyzed skin wound healing using MFG-E8 wild-type (WT) and KO mice. We demonstrate that MFG-E8 production was increased and that MFG-E8 accumulated in granulation tissue during wound healing, and that wound healing in MFG-E8 KO mice was delayed. We relate delayed wound healing to diminished angiogenesis and myofibroblast infiltration in wounds in MFG-E8 KO mice.     

Full‐Arch Implant‐Supported Fixed Dental Prosthesis Retained by a Combination of Galvano‐Telescopic Copings and Screws: A Clinical Report

Full‐arch screw‐retained implant‐supported fixed dental prostheses have a high long‐term success rate and are considered the gold standard by many clinicians. However, accurate fabrication of a passive fit long‐span prosthesis can be challenging. A novel intraoral adhesion method using galvano‐telescopic copings was proposed as a way of improving prosthetic fit for edentulous patients. This report describes the treatment of a 74‐year‐old female with a full‐arch implant‐supported dental prosthesis, supported by a combination of galvano‐telescopic copings and screws to prevent retention loss. Four years have passed since this superstructure was placed, during this time she exhibited a good clinical course with no inflammation noted in surrounding tissues. Treatment with an implant‐supported fixed dental prosthesis, retained by a combination of galvano‐telescopic copings and screws, can be a useful alternative treatment for edentulous patients.     

Influence of Dialysis Duration on the Outcome of Living Kidney Transplantation

Previous studies have reported negative impacts of long‐term dialysis on kidney transplantation (KTx) outcomes. However, advances in surgical techniques, immunosuppressive therapies, and post‐transplant monitoring have led to an impressive increase in patient and allograft survival. Thus, the number of KTx among patients on long‐term dialysis is increasing. We evaluated the influence of dialysis duration on the outcome of living donor KTx. Between January 2000 and October 2011, we performed 1098 first KTx from living donors in adults (>18 years). We divided the patients into six groups, A group: pre‐emptive kidney transplantation, B group: <24 months duration of dialysis, C group: 25–60 months duration, D group: 61–120 months duration, E group: 121–240 months duration, and F group: ≥241 months duration. The 5‐year patient survival rates were 95.7, 98.8, 99.0, 99.0, 97.3, and 100% in groups A–F, respectively. The 5‐year graft survival rates were 91.3, 95.6, 94.2, 96.3, 90.7, and 100% in groups A–F, respectively. No significant differences were observed in patient or graft survival among the six groups. Longer dialysis duration was correlated with lower rates of preoperative hypertension and diabetes mellitus. Survivors of long‐term dialysis tended to be in good compliance with self‐management. If recipients of living KTx have few complications, good prognoses are expectable even if dialysis periods are very long.     

Skeletal Muscle Loss Is Negatively Associated With Single‐Pool Kt/V and Dialysis Duration in Hemodialysis Patients

We evaluated the skeletal muscle loss in hemodialysis (HD) patients by bioelectrical impedance analysis (BIA) and handgrip strength test. Thirty‐four HD patients and 16 healthy subjects (control group) were measured for skeletal muscle mass normalized as the skeletal muscle mass index (SMI), calculated as skeletal muscle mass (kg)/height (m)² using a tetrapolar bioelectrical impedance plethysmograph. Handgrip strength test was also performed using a hand dynamometer in both groups. In HD patients, the associations of SMI and handgrip strength with age, sex, HD conditions, and HD parameters such as body mass index (BMI), single‐pool Kt/V (spKt/V), normalized protein catabolic rate (nPCR), creatinine generation rate (CGR) and serum albumin level (Alb) were investigated. SMI of HD patients (4.58 ± 0.95 kg/m²) was significantly lower than that of the control group (5.55 ± 0.80 kg/m², P < 0.01). The handgrip strength of HD patients (19.9 ± 7.74 kg) was also significantly lower than that of the control group (33.0 ± 8.94 kg, P < 0.01). In HD patients, HD duration was associated with both SMI and handgrip strength. Among HD parameters, spKt/V was negatively associated with both SMI and handgrip strength, BMI and Alb were positively associated with SMI, while nPCR and CGR were associated with neither SMI nor handgrip strength. HD duration independently contributed to skeletal muscle loss and the value of spKt/V may be affected by skeletal muscle loss in HD patients.     

Fast rebinding increases dwell time of Src homology 2 (SH2)-containing proteins near the plasma membrane

Receptor tyrosine kinases (RTKs) control a host of biological functions by phosphorylating tyrosine residues of intracellular proteins upon extracellular ligand binding. The phosphotyrosines (p-Tyr) then recruit a subset of ～100 Src homology 2 (SH2) domain-containing proteins to the cell membrane. The in vivo kinetics of this process are not well understood. Here we use total internal reflection (TIR) microscopy and single-molecule imaging to monitor interactions between SH2 modules and p-Tyr sites near the cell membrane. We found that the dwell time of SH2 modules within the TIR illumination field is significantly longer than predictions based on chemical dissociation rate constants, suggesting that SH2 modules quickly rebind to nearby p-Tyr sites after dissociation. We also found that, consistent with the rebinding model, the effective diffusion constant is negatively correlated with the respective dwell time for different SH2 domains and the dwell time is positively correlated with the local density of RTK phosphorylation. These results suggest a mechanism whereby signal output can be regulated through the spatial organization of multiple binding sites, which will prompt reevaluation of many aspects of RTK signaling, such as signaling specificity, mechanisms of spatial control, and noise suppression.     

Ameloblastoma, desmoplastic type: a case report with characteristic radiological presentation

Ameloblastoma, desmoplastic type, is a rare lesion for which radiographic images are even less common, and such lesions are sometimes considered to be variant types. It is defined as a variant of ameloblastoma with specific imaging and histological features. This lesion occurs with the same frequency in the maxilla and mandible, although the predominant site is the anterior-premolar site in both the mandible and maxilla. For our case of ameloblastoma, desmoplastic type, resected from the right anterior to premolar maxilla, the radiographic appearance and histopathological findings were compared. Computed tomography images revealed that the lesion had a multilocular structure with many smaller septa at its periphery. Although expansion toward the maxillary sinus was suggested radiologically, invasion of the mucosa into the floor of the maxillary sinus was found on histopathological examination.     

Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia

Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.