MiR-760 targets HBEGF to control cartilage extracellular matrix degradation in osteoarthritis.

The present study was developed to explore whether microRNA (miR)-760 targets heparin-binding EGF-like growth factor (HBEGF) to control cartilage extracellular matrix degradation in osteoarthritis. Both miR-760 and HBEGF expression levels were analysed in human degenerative cartilage tissues and in interleukin (IL)-1ß/tumour necrosis factor (TNF)-α-treated chondrocytes in vitro. A series of knockdown and overexpression assays were then used to gauge the functional importance of miR-760 and HBEGF in OA, with qPCR and western immunoblotting analyses. Bioinformatics assays were used to identify putative miR-760 target genes, with these predictions then being validated through RNA pulldown and luciferase reporter assays. A murine anterior cruciate ligament transection model of OA was then established to prove the in vivo relevance of these findings. These experiments revealed that human degenerative cartilage tissues exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF levels. IL-1ß/TNF-α-treated chondrocytes also exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF expression. When chondrocytes were transfected with either miR-760 inhibitor or HBEGF overexpression constructs, this was sufficient to interfere with degradation of the extracellular matrix (ECM). Moreover, miR-760 was confirmed to control chondrocyte matrix homeostasis by targeting HBEGF, and the overexpression of HBEGF partially reversed the effects of miR-760 mimic treatment on the degradation of the cartilage ECM. When OA model mice were administered an intra-articular knee injection of an adenoviral vector encoding a miR-760 mimic construct, cartilage ECM degradation was aggravated. Conversely, the overexpression of HBEGF in OA model mice partially reversed the effects of miR-760 overexpression, restoring appropriate ECM homeostasis. In summary, these data indicated that the miR-760/HBEGF axis plays a central role in orchestrating the pathogenesis of OA, making it a candidate target for therapeutic efforts in OA.

Heparin-binding EGF-like growth factor attenuates lung inflammation and injury in a murine model of pulmonary emphysema.

Pulmonary inflammation and progressive lung destruction are the major causes of chronic obstructive pulmonary disease (COPD), resulting in emphysema and irreversible pulmonary dysfunction. Heparin-binding EGF-like growth factor (HB-EGF), is known to play a protective role in the process of various inflammatory diseases. However, its effect on COPD is poorly understood. This study was designed to determine the effect of HB-EGF on lung inflammation and injury in a murine model of pulmonary emphysema. HB-EGF promoted percent survival and body weight, attenuated lung injury, inflammatory cells, and cytokines infiltration, and prevented lung function decline. Additionally, treatment of rHB-EGF suppressed the nuclear translocation of nuclear factor κB (NF-κB)/p65, decreased TUNEL-positive cells and the expression of caspase 3, and increased the expression of PCNA, HB-EGF, and EGF receptor (EGFR). We conclude that HB-EGF attenuates lung inflammation and injury, probably through the activation of EGFR, followed by suppression of NF-ΚB signalling, promotion of cell proliferation, and inhibition of apoptosis.

Epithelial separation theory for post-tonsillectomy secondary hemorrhage: evidence in a mouse model and potential heparin-binding epidermal growth factor-like growth factor therapy.

OBJECTIVE: To provide histological evidence to investigate a theory for post-tonsillectomy secondary hemorrhage (PTH) in a mouse model and to evaluate the potential for heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment on wound healing in this model. METHODS: A prospective randomized single-blinded cohort study. A uniform tongue wound was created in 84 mice (day 0). Mice were randomized to HB-EGF (treatment, n = 42) or saline (control, n = 42). In treatment mice, HB-EGF 5 µg/ml was administered intramuscularly into the wound daily (days 0-14). In control mice, normal saline was administered daily. Three mice from each group were sacrificed daily through day 14 and the wounds evaluated histologically by blinded reviewers. RESULTS: Key stages of wound healing, including keratinocyte proliferation and migration, wound contraction, epithelial separation, and neoangiogenesis, are defined with implications for post-tonsillectomy wound healing. Epithelial separation (59 vs. 100%, p = 0.003) and wound reopening (8 vs. 48%, p < 0.001) were reduced with HB-EGF. Epithelial thickness (220 vs. 30 µm, p = 0.04) was greater with HB-EGF. Wound closure (days 4-5 vs. day 6, p = 0.01) occurred earlier with HB-EGF. CONCLUSIONS: In healing of oral keratinocytes on muscle epithelial separation secondary to muscle, contraction occurs concurrently with neoangiogenesis in the base of the wound, increasing the risk of hemorrhage. This potentially explains why post-tonsillectomy secondary hemorrhage occurs and its timing. HB-EGF-treated wounds showed greater epithelial thickness, less frequent epithelial separation and wound reopening, and earlier wound closure prior to neovascularization, suggesting that HB-EGF may be a potential preventative therapy for PTH. LEVEL OF EVIDENCE: NA-animal studies or basic research.

Heparin Binding Epidermal Growth Factor-Like Growth Factor Heals Chronic Tympanic Membrane Perforations With Advantage Over Fibroblast Growth Factor 2 and Epidermal Growth Factor in an Animal Model.

HYPOTHESIS: That heparin binding epidermal growth factor-like growth factor (HB-EGF) heals chronic tympanic membrane (TM) perforations at higher rates than fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) in an animal model. BACKGROUND: A nonsurgical treatment for chronic TM perforation would benefit those unable to access surgery or those unable to have surgery, as well as reducing the cost of tympanoplasty. Growth factor (GF) treatments have been reported in the literature with variable success with the lack of a suitable animal providing a major obstacle. METHODS: The GFs were tested in a validated mouse model of chronic TM perforation. A bioabsorbable hydrogel polymer was used to deliver the GF at a steady concentration as it dissolved over 4 weeks. A control (polymer only, n = 18) was compared to polymer loaded with HB-EGF (5 µg/ml, n = 18), FGF2 (100 µg/ml, n = 19), and EGF (250 µg/ml, n = 19). Perforations were inspected at 4 weeks. RESULTS: The healing rates, as defined as 100% perforation closure, were control (5/18, 27.8%), HB-EGF (15/18, 83.3%), FGF2 (6/19, 31.6%), and EGF (3/19, 15.8%). There were no differences between FGF2 (p = 0.80) and EGF (p = 0.31) with control healing rates. HB-EGF (p = 0.000001) showed a significant difference for healing. The HB-EGF healed TMs showed layers similar to a normal TM, whereas the other groups showed a lack of epithelial migration. CONCLUSION: This study confirms the advantage of HB-EGF over two other commonly used growth factors and is a promising nonsurgical treatment of chronic TM perforations.

M1 to M2 macrophage polarization in heparin-binding epidermal growth factor-like growth factor therapy for necrotizing enterocolitis.

BACKGROUND: Macrophages can be polarized into proinflammatory (M1) and anti-inflammatory (M2) subtypes. However, whether macrophage polarization plays a role in necrotizing enterocolitis (NEC) remains unknown. MATERIALS AND METHODS: Macrophages were derived from the THP-1 human monocyte cell line. Apoptosis of human fetal small intestinal epithelial FHs-74 cells was determined by Annexin V/propidium iodide flow cytometry and by Western blotting to detect cleaved caspase-3. The effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on macrophage polarization was determined by flow cytometry with M1/M2 markers and real time polymerase chain reaction. In vivo, experimental NEC was induced in mouse pups by repeated exposure to hypoxia, hypothermia, and hypertonic feedings. Intestinal histologic sections were subjected to immunohistochemical staining for the detection of M1 and M2 macrophages. RESULTS: In vitro, FHs-74 cell apoptosis was increased after coculture with macrophages and lipopolysaccharide (LPS). This apoptosis was increased by exposure to M1-conditioned medium and suppressed by exposure to M2-conditioned medium. HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via signal transducers and activators of transcription 3 activation. Addition of HB-EGF to LPS-stimulated macrophages suppressed the proapoptotic effects of the macrophages on FHs-74 cells. In vivo, we found enhanced intestinal macrophage infiltration in pups subjected to NEC, most of which were M1 macrophages. HB-EGF treatment of pups subjected to experimental NEC significantly reduced M1 and increased M2 polarization and protected the intestines from NEC. CONCLUSIONS: M1 macrophages promote NEC by increasing intestinal epithelial apoptosis. HB-EGF protects the intestines from NEC by preventing M1 and promoting M2 polarization.

A pain in the NEC: research challenges and opportunities.

This lecture will describe how the properties of heparin-binding EFG-like growth factor (HB-EGF) can be utilized to protect the intestines from necrotizing enterocolitis (NEC), to augment the effects of stem cells in the treatment of NEC, and to improve the production of tissue-engineered intestine. It will also explore the role of the enteric nervous system in the development of NEC.

Heparin binding-epidermal growth factor-like growth factor for the regeneration of chronic tympanic membrane perforations in mice.

We aim to explore the role of epidermal growth factor (EGF) ligand shedding in tympanic membrane wound healing and to investigate the translation of its modulation in tissue engineering of chronic tympanic membrane perforations. Chronic suppurative otitis media (CSOM) is an infected chronic tympanic membrane perforation. Up to 200 million suffer from its associated hearing loss and it is the most common cause of pediatric hearing loss in developing countries. There is a need for nonsurgical treatment due to a worldwide lack of resources. In this study, we show that EGF ligand shedding is essential for tympanic membrane healing as it's inhibition, with KB-R7785, leads to chronic perforation in 87.9% (n=58) compared with 0% (n=20) of controls. We then show that heparin binding-EGF-like growth factor (5 µg/mL), which acts to shed EGF ligands, can regenerate chronic perforations in mouse models with 92% (22 of 24) compared with 38% (10 of 26), also with eustachian tube occlusion with 94% (18 of 19) compared with 9% (2 of 23) and with CSOM 100% (16 of 16) compared with 41% (7 of 17). We also show the nonototoxicity of this treatment and its hydrogel delivery vehicle. This provides preliminary data for a clinical trial where it could be delivered by nonspecialist trained healthcare workers and fulfill the clinical need for a nonsurgical treatment for chronic tympanic membrane perforation and CSOM.