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- Almqvist, Sofia, 1980, et al.
(författare)
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Amelogenin is phagocytized and induces changes in integrin configuration, gene expression and proliferation of cultured human dermal fibroblasts
- 2010
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Ingår i: Journal of Materials Science. Materials in Medicine. - : Springer Science and Business Media LLC. - 1573-4838 .- 0957-4530. ; 21:3, s. 947-954
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Tidskriftsartikel (refereegranskat)abstract
- Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100 and 1000 μg/ml increased binding of NHDF via several integrins, including αvβ3, αvβ5 and α5β1. Further, both surface interaction and cellular uptake of amelogenin by NHDF was observed using scanning and transmission electron microscopy. Gene microarray studies showed >8-fold up or down-regulation of genes, of which most are involved in cellular growth, migration and differentiation. The effect of amelogenin was exemplified by increased proliferation over 7 days. In conclusion, the beneficial effects of amelogenin on wound healing are possibly conducted by stimulating fibroblast signalling, proliferation and migration via integrin interactions. It is hypothesized that amelogenin stimulates wound healing by providing connective tissue cells with a temporary extracellular matrix.
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| 2. |
- Almqvist, Sofia, 1980, et al.
(författare)
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Amelogenins modulate cytokine expression in LPS-challenged cultured human macrophages.
- 2012
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Ingår i: Cytokine. - : Elsevier BV. - 1096-0023 .- 1043-4666. ; 58:2, s. 274-9
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Tidskriftsartikel (refereegranskat)abstract
- Amelogenins are enamel matrix proteins with a proven ability to restore tissues in patients with advanced periodontitis and chronic skin wounds. To explore the mechanisms of action of amelogenins in wound inflammation, the in vitro effect on the expression of selected cell mediators involved in inflammation and tissue repair from human monocyte-derived macrophages was studied. Macrophages were treated with amelogenins in serum-enriched medium with simultaneous lipopolysaccharide (LPS) stimulation, for 6, 24 and 72 h, and the conditioned culture medium was analysed for 28 different cytokines. Amelogenin treatment directed the LPS-induced release of both pro- and anti-inflammatory cytokines towards an alternatively activated macrophage phenotype. This change in activation was also demonstrated by the amelogenin-induced secretion of alternative macrophage activation-associated CC chemokine-1 (AMAC-1, also known as CCL18; p<0.001), a well-documented marker of alternative activation. Amelogenins were also shown significantly to increase the macrophage expression of vascular endothelial growth factor and, to a lesser but significant extent, insulin-like growth factor-1 after 24h of culture. The results of the present in vitro study show that monocyte-derived macrophages stimulated by inflammatory agonist LPS respond to the treatment with amelogenins by reducing the pro-inflammatory activity and increasing the expression of tissue repair mediators.
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| 4. |
- Almqvist, Sofia, 1980, et al.
(författare)
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Amelogenins promote an alternatively activated macrophage phenotype in vitro.
- 2011
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Ingår i: International Journal of Nano and Biomaterials (IJNBM). - 1752-8933. ; 3:3, s. 282-298
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Amelogenins are extracellular matrix proteins used for the topical treatment of chronically inflamed tissues. The influence of amelogenins on human monocyte-derived macrophages was studied by measuring the concentrations of cytokines in culture supernatants. The interactions of cells and protein aggregates were visualised by transmission electron microscopy. The amelogenin treatment of macrophages increased several pro- and anti-inflammatory cytokines, including alternative macrophage activation marker AMAC-1 (p < 0.001) and vascular endothelial growth factor (VEGF; p < 0.001). The levels were independent of cytochalasin B, although amelogenin aggregates were ingested by macrophages. Amelogenin effect was compared with that of tyrosine-rich amelogenin peptide, which apart from augmented VEGF levels (p < 0.05), had no significant influence on the other cytokines analysed. In conclusion, amelogenins increased the macrophage release of key cell mediators involved in tissue repair. The effect was independent of phagocytosis, implying a receptor-mediated signal. The markedly increased levels of AMAC-1 suggest that amelogenins promote a reparative macrophage phenotype.
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| 9. |
- Almqvist, Sofia, 1980, et al.
(författare)
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Ex Vivo Study of the Angiogenic Effect of the Extracellular Matrix Protein Amelogenin
- 2008
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Ingår i: Abstract, The 9th New Jersey Symposium on Biomaterials Science and regenerative medicine, New Jersey, USA. ; 29-31 October
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Konferensbidrag (refereegranskat)abstract
- Introduction: Angiogenesis is crucial for wound healing but is often impaired in chronic wounds. The process is dependent on the interaction of endothelial cells and the extracellular matrix (ECM), which is mediated by cell membrane integrins. Amelogenin is an extracellular matrix protein that has been reported to promote formation of granulation tissue and repair of chronic venous leg ulcers and elevate the pro-angiogenic vascular endothelial growth factor in dermal fibroblasts.1-3 This study investigated the effect of amelogenin on angiogenesis in an ex vivo sprouting assay and related the findings to the cell surface integrin expression. Methods: Chick aortic arch assay: Transverse sections of the aortic arch of 13-day-old chick embryos were attached and sealed with Matrigel to the bottom of a 48-well plate. Amelogenin was added (0.01 mg/ml, 0.1 mg/ml and 1 mg/ml) in serum-free endothelial basal growth medium. Porcine serum albumin was used as control for unspecific protein effects. The plates were incubated at 37°C and sprouting was assessed at 24 h and 48 h by microscopy and scored from 0 to 6 (arbitrary units) by a blinded observer. Integrin assay: Human dermal microvascular endothelial cells (Promocell) were seeded in complete cell growth medium alone or supplemented with 0.1 mg/ml, 1 mg/ml amelogenin or 20 µg/ml fibronectin. After 24 h incubation in 37°C, cells were gently harvested with the non-enzymatic buffer (EDTA/PBS). Upregulated integrins/subunits were detected by an Integrin-Mediated Cell Adhesion Array (Chemicon), where cells expressing specific integrins (α1, α2, α3, α4, α5, αv, β1, β2, β3, β4, β6, αvβ3, αvβ5 and α5b1) are captured by surface immobilized antibodies. Results and Discussion: Amelogenin at 0.1 mg/ml significantly (p = 0.001) increased micro-vessel outgrowth by 76 % from the explants compared with control explants after 48 h of incubation. No significant sprouting was observed with the non-specific protein control porcine serum albumin or medium only. The preliminary data from the integrin assay show that amelogenin at 0.1 mg/ml also displays a broad up-regulation of several integrins/subunits. This result is comparable to the positive control, fibronectin, an ECM protein involved in all phases of tissue repair. Taken together, the present observations suggest that the angiogenic effects might be explained by the cell binding properties of amelogenin. Conclusions: Amelogenin stimulated micro-vessel outgrowth in the chick aortic arch assay possibly through up-regulation of several integrins and subunits important for cell interaction with the ECM. The pro-angiogenic property may contribute to the beneficial effects reported after treatment of chronic ulcers with the novel ECM therapy containing amelogenin. Acknowledgements: The studies were supported by the Swedish Research Council (grant K2006-73X-09495-16-3), Mölnlycke Health Care AB, the VINNOVA VinnVäxt Program Biomedical Development in Western Sweden, and the Danish Medical Research Council (22-02-0287). References: 1. Mirastschijski U. et al. (2004) Wound Repair Regen. 12:100-108. 2. Vowden P. et al. (2006) Wound Repair Regen. 14:240-248. 3. Ågren M. S. et al. (2007) Wound Repair Regen. 15:A139.
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| 10. |
- Almqvist, Sofia, 1980, et al.
(författare)
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In Vitro Effect of Amelogenin on Selected Cell Mediators from Human Monocytes
- 2008
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Ingår i: 8th World Biomaterials Congress, Amsterdam, The Netherlands.
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Konferensbidrag (refereegranskat)abstract
- Introduction: Inflammation is an integral part of the normal wound healing response. Besides clearing the wound of invading microbes and debris, inflammatory cells are believed to be crucial coordinators of the repair process, acting both as phagocytes and as a major source of growth factors and other signals [1]. In non-healing skin ulcers the repair process is stuck in the inflammation phase [2]. Excessive inflammation can reflect an imbalance in the transformation of phenotype between the classically activated, inflammatory macrophage and the alternatively activated macrophage involved in immunosuppression and tissue repair [3]. Amelogenin is a hydrophobic extracellular matrix protein that under physiological conditions will self assemble into nanospheres which in turn may form larger aggregates. Treatment with amelogenin has shown enhanced skin wound healing in an in vivo study in rabbits [4]. In addition, amelogenin has been proposed to have anti-inflammatory properties by attenuation of lipopolysaccharide (LPS)- and peptidoglucan-induced production of selected pro-inflammatory cytokines by human blood cells [5]. The present study was initiated to determine the effects of amelogenin on human monocyte secretion of factors which modulate both inflammation and tissue repair. Materials and Methods: Lyophilized amelogenin from Biora AB (Malmö, Sweden) was dissolved in 17 mM acetic acid. Human monocytes were obtained from six healthy blood donors by isolation using the separation gradient PercollTM in two steps according to Pertoft et al. [6]. The isolated monocytes were cultured for 24 h at 37ºC with 5% CO2 and 95% humidity. Thereafter the supernatants and non-adherent cells were removed. Fresh medium (RPMI, 5% foetal bovine serum, antibiotics) containing amelogenin, 0, 0.01, 0.1 and 1.0 mg/ml, and with or without addition of LPS, was added to the wells in triplicates. The plates were again incubated for 24 h. The supernatants were analyzed with commercial human ELISA assays for tumour necrosis factor- (TNF-), interleukin-10 (IL-10), macrophage inflammatory protein-1 (MIP-1), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and insulin like growth factor-1 (IGF-1). Results: Amelogenin treatment markedly altered the expression of factors by human monocytes. Amelogenin significantly reduced LPS-induced TNF- secretion, whereas the IL-10 expression was increased. Monocyte secretion of the two inflammatory chemokines MIP-1 and MCP-1 (Figure; mean ± SEM, n=6) was also affected by amelogenin treatment. Furthermore, amelogenin significantly increased monocyte secretion of VEGF (Figure; mean ± SEM, n=6) and IGF-1, although to a lesser extent, after 24 h culture. Conclusions: The amelogenin effects correlate to protein concentration, however not in a dose dependent manner, but instead the cell responses may reflect a concentration related difference in self assembly of the amelogenin protein. The observed changes in cytokine and chemokine expression are markedly affected by simultaneous LPS-induced inflammation activation, revealing possible anti-inflammatory properties of the amelogenin protein. In addition, the several-fold increase in VEGF-levels by monocytes provides a possible mechanism for the observed pro-angiogenic effect in vivo [4]. These in vitro results indicate that the extracellular matrix protein amelogenin by virtue of its interaction with human monocytes may modulate inflammation and tissue repair. Acknowledgements: The support from the Swedish Research Council (grant K2006-73X-09495-16-3), Mölnlycke Health Care Group AB and the VINNOVA VinnVäxt Program Biomedical Development in Western Sweden, is gratefully acknowledged. References: 1. Martin, P., et al. Trends Cell Biol., 15, 599, 2005. 2. Ågren, M.S., et al. Acta Derm Venereol Suppl (Stockh). 210, 3, 2000. 3. Duffield, J.S. Clin Sci (Lond), 104, 27, 2003 4. Mirastschijski, U., et al. Wound Repair Regen., 12, 100, 2004. 5. Myhre, A.E., et al. J Periodontal Res., 41, 208, 2006. 6. Pertoft, H., et al. J Immunol Methods., 33, 221, 1980.
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