1. Complement factor C5a as mast cell activator mediates vascular remodelling in vein graft disease
Peter J van Santbrink,Hans W M Niessen,Anouk Wezel,Trent M Woodruff,Abbey Schepers,Paul H A Quax,Ilze Bot,Johan Kuiper,Jaap F Hamming,Margreet R de Vries Cardiovasc Res . 2013 Feb 1;97(2):311-20. doi: 10.1093/cvr/cvs312.
Aims:Failure of vein graft conduits due to vein graft thickening, accelerated atherosclerosis, and subsequent plaque rupture is applicable to 50% of all vein grafts within 10 years. New potential therapeutic targets to treat vein graft disease may be found in components of the innate immune system, such as mast cells and complement factors, which are known to be involved in atherosclerosis and plaque destabilization. Interestingly, mast cells can be activated by complement factor C5a and, therefore, a direct role for C5a-mediated mast cell activation in vein graft disease is anticipated. We hypothesize that C5a-mediated mast cell activation is involved in the development and destabilization of vein graft lesions.Methods and results:Mast cells accumulated in time in murine vein graft lesions, and C5a and C5a-receptor (CD88) expression was up-regulated during vein graft disease in apolipoprotein E-deficient mice. Mast cell activation with dinitrophenyl resulted in a profound increase in vein graft thickening and in the number of plaque disruptions. C5a application enhanced vein graft lesion formation, while treatment with a C5a-receptor antagonist resulted in decreased vein graft disease. C5a most likely exerts its function via mast cell activation since the mast cell inhibitor cromolyn totally blocked C5a-enhanced vein graft disease.Conclusion:These data provide evidence that complement factor C5a-induced mast cell activation is highly involved in vein graft disease, which identifies new targets to prevent vein graft disease.
2. Development and validation of a LC-MS/MS assay for pharmacokinetic studies of complement C5a receptor antagonists PMX53 and PMX205 in mice
John D Lee,Richard J Clark,Trent M Woodruff,Vinod Kumar Sci Rep . 2018 May 25;8(1):8101. doi: 10.1038/s41598-018-26387-4.
PMX53 and PMX205 are cyclic hexapeptide inhibitors of complement C5a receptors (C5aR1), that are widely used to study C5aR1 pathobiology in mouse models of disease. Despite their widespread use, limited information regarding their pharmacokinetics have been reported. Here, a bioanalytical method for the quantitative determination of PMX53 and PMX205 in plasma, brain and spinal cord of mice was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. The LC-MS/MS method was validated in all three matrices according to regulatory guidelines and successfully applied to pharmacokinetic studies of PMX53 and PMX205 in C57BL/6 J mice following intravenous administration. The developed method was highly sensitive and sufficiently accurate with a lower limit of quantification within the range of 3-6 ng/ml in extracted plasma samples and 3-6 ng/g in processed tissue samples, which outperforms previously published LC-MS/MS methods. The results thus support the suitability, reliability, reproducibility and sensitivity of this validated technique. This method can therefore be applied to perform a complete pre-clinical investigation of PMX53 and PMX205 pharmacokinetics in mice.
3. Expression of complement C5a receptor and the viability of 4T1 tumor cells following agonist-antagonist treatment
Mohd Hezmee Mohd Noor,Mohd Mokrish Md Ajat,Norhaifa Ganti,Intan Shameha Abdul Razak,Abdul Rahman Omar,Nurneqman Nashreq Kosni J Cancer Res Ther . 2016 Apr-Jun;12(2):590-6. doi: 10.4103/0973-1482.146066.
Background:Complement system is theoretically believed to halt the progression of tumor by the activity of C5a/CD88. Protein C5a is a potent pro.inflammatory mediator that activates the complement system by binding to its receptor.Objectives:The purpose of this study is to determine the expression of the anaphylatoxin C5a receptor on 4T1 cell line and to study the viability of the cells after being treated with the C5a peptides.Materials and methods:The cells 4T1 had undergone immunofluorescence staining, conventional polymerase chain reaction (PCR) and real-time PCR for the expression of determination part. Whereas Alamar Blue and MTT assays were conducted for the viability study of the cells.Results:The cells showed positive result in expressing the receptor of the C5a through immunostaining and PCR. The CT value recorded at initial dilution was 22.24. In cell viability assay, the cell was treated with C5a peptides, PMX205 and EP54. The purpose of this treatment was to see whether C5a had a direct effect on the cell itself using both assays. The result showed that PMX205, which is an antagonist, gave more effects towards the cell as compared with the treatment of EP54.Conclusion:This experiment shows the presence of C5a receptor on 4T1 cell line. We believe that the antagonist peptide is eligible to be used widely in cancer immunotherapy field; but in vivo studies need to be carried out first in the future, as it will determine how these drugs affect the tumor cell growth.
4. The Alternative Receptor for Complement Component 5a, C5aR2, Conveys Neuroprotection in Traumatic Spinal Cord Injury
Trent M Woodruff,Marc J Ruitenberg,Stephen M Taylor,Faith H Brennan,Patrick J C Biggins J Neurotrauma . 2017 Jun 15;34(12):2075-2085. doi: 10.1089/neu.2016.4701.
This study investigated the role of the alternative receptor for complement activation fragment C5a, C5aR2, in secondary inflammatory pathology after contusive spinal cord injury (SCI) in mice. C5ar2-/-mice exhibited decreased intraparenchymal tumor necrosis factor alpha and interleukin-6 acutely post-injury, but these reductions did not translate into improved outcomes. We show that loss of C5aR2 leads to increased lesion volumes, reduced myelin sparing, and significantly worsened recovery from SCI in C5ar2-/-animals compared to wild-type (WT) controls. Loss of C5aR2 did not alter leukocyte mobilization from the bone marrow in response to SCI, and neutrophil recruitment/presence at the lesion site was also not different between genotypes. Acute treatment of SCI mice with the selective C5aR1 antagonist, PMX205, improved SCI outcomes, compared to vehicle controls, and, importantly, fully alleviated the worsened recovery of C5ar2-/-mice compared to their WT counterparts. Collectively, these findings indicate that C5aR2 is neuroprotective and a novel target to restrain injurious C5a signaling after a major neurotraumatic event.