title: Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts.
abstract: BACKGROUND
  Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2-ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2).
  
  
  METHODS
  Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors.
  
  
  RESULTS
  The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells.
  
  
  CONCLUSIONS
  These results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2-ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing.
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: mtohidnezhad@ukaachen.de.
authors.forename: Mersedeh
authors.initials: M
authors.lastname: Tohidnezhad
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: cwruck@ukaachen.de.
authors.forename: Christoph-Jan
authors.initials: CJ
authors.lastname: Wruck
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: aslowik@ukaachen.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Slowik
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: nkweider@ukaachen.de.
authors.forename: Nisreen
authors.initials: N
authors.lastname: Kweider
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: rbeckmann@ukaachen.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Beckmann
authors.affiliation: Department of Trauma Surgery, University Hospital of Schleswig Holstein, Campus Kiel, Arnold-Heller Str 3, D-24105 Kiel, Germany. Electronic address: andreasbayer-kiel@web.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bayer
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: astrid.houben@ukmuenster.de.
authors.forename: Astrid
authors.initials: A
authors.lastname: Houben
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: lbrandenburg@ukaachen.de.
authors.forename: Lars-Ove
authors.initials: LO
authors.lastname: Brandenburg
authors.affiliation: Department of Trauma Surgery, University Hospital of Schleswig Holstein, Campus Kiel, Arnold-Heller Str 3, D-24105 Kiel, Germany. Electronic address: deike.varoga@uksh-kiel.de.
authors.forename: Deike
authors.initials: D
authors.lastname: Varoga
authors.affiliation: Department of Oral and Maxillofacial Surgery, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany. Electronic address: tsoenmez@ukaachen.de.
authors.forename: Tolga-Taha
authors.initials: TT
authors.lastname: Sönmez
authors.affiliation: Institute of General Mechanics, RWTH Aachen University, D-52062 Aachen, Germany. Electronic address: Stoffel@iam.rwth-aachen.de.
authors.forename: Marcus
authors.initials: M
authors.lastname: Stoffel
authors.affiliation: Department of Orthopaedic Surgery, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany. Electronic address: hjahr@ukaachen.de.
authors.forename: Holger
authors.initials: H
authors.lastname: Jahr
authors.affiliation: Department of Trauma Surgery, University Hospital of Schleswig Holstein, Campus Kiel, Arnold-Heller Str 3, D-24105 Kiel, Germany. Electronic address: sebastian.lippross@uksh-kiel.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lippross
authors.affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. Electronic address: tpufe@ukaachen.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Pufe
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D045744:Cell Line, Tumor; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D010006:Osteoblasts / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D036341:Intercellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species
keywords: Fracture healing; Nrf2; PRP; VEGF