ProteaseActivated Receptors: Their Role in the Development of Inflammation

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Protease-Activated ReceptorsTheir Role in the
Development of Inflammation

• Aaron Hirschfeld
• Department of Biological Sciences

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Protease-Activated Receptors

• Protease-activated receptors (PARs) have been
  associated with inflammation and neurogenic
  diseases.
• PARs are a subfamily of the G-protein coupled
  receptors.
• PAR1-PAR4 have been identified, with PAR1 and
  PAR2 being the most studied.

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PARs Activation

• PARs are activated by a mechanism where the
  N-terminal sequence is cleaved, unmasking a
  tethered ligand that binds to the second exoloop,
  thereby activating the receptor.

PAR1
PAR2
N
N
C
C
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PARs Activation

• PARs are activated by a mechanism where the
  N-terminal sequence is cleaved, unmasking a
  tethered ligand that binds to the second exoloop,
  thereby activating the receptor.

PAR1
PAR2
N
N
C
C
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A Rat Model for Inflammation

• The rat prostate has been used successfully to
  model bacterial induced inflammation.
• Development of a PAR mediated inflammatory
  response in the prostate could provide a
  foundation for theories relating to PAR
  activation during joint injury, inflammation and
  trauma.
• Bacterial infection of prosthetics and subsequent
  biofilm formation could result in exogenous
  protease production and PAR activation in joints.

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Hypothesis

• Activation of protease-activated receptors will
  result in inflammation in the prostate, observed
  by using the rat model for prostatitis developed
  by the Ceri lab.

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Research Objectives

• Use immunohistochemical techniques to localize
  PAR2 in the rat prostate tissue.
• Induce inflammation in the rat prostate via a
  PAR2 mediated pathway.
• Observe the inflammatory changes over a period of
  48 hours.

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Immunohistochemistry

• In order to localize PAR2 in the rat prostate the
  B5 and SLAW-A antibodies to PAR2, obtained from
  Dr. Morley Hollenberg, were used.

N
C
9
B5 Antibody
B5
B5 peptide
No B5
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Activated PAR2 and Inflammation
Inoculation of the prostate via catheter
TRYPSIN
E. Coli CP9
PBS
Harvest the tissue at 6 hrs., 12 hrs., 24 hrs.,
and 48 hrs.
Examine for gross morphology and histology
Homogenize and save tissue supernatent for future
cytokine analysis
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Prostate Histology
Edematous stromal tissue
Minimal stromal tissue
PBS Inoculated Rat Prostate (12 hrs.)
Trypsin Inoculated Rat Prostate (12 hrs.)
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Prostate Histology
Thickened acini, ill-defined borders and
neutrophil infiltration
Well-defined epithelial borders
PBS Inoculated Rat Prostate (48 hrs.)
Trypsin (200U) Inoculated Rat Prostate (24hrs.)
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Gross Morphological Score
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Histological Score
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Conclusions

• PAR2 has been found at the membrane surface of
  the acini in the rat prostate, but association
  with neurons is inconclusive at this time.
• Treatment with trypsin leads to the development
  of prostatitis in the rat.
• Different concentrations of the protease trypsin,
  50U or 200U, result in different inflammatory
  profiles over 48 hours.

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Future Directions

• Observe inflammation over a longer time with 50U
  of trypsin.
• Use immunohistochemistry to localize PAR2 to
  neurons.
• Use agonist peptides to specifically induce
  inflammation via PARs.
• Use an neurokinin receptor-1 antagonist to assess
  the neurogenic mechanisms of inflammation and
  PAR2 activation.
• Extend this model to various joints in the rat.

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Acknowledgements

• Markin-Flanagan Studentship
• The Ceri Lab
• Dr. Howard Ceri
• Carol Stremick, Kerry Tomlin, Bill Huddleston,
  Lorine Pelly, Van Phan
• The Hollenberg Lab
• Dr. Morley Hollenberg
• Suranga Wijesuriya
• The Biofilm Research Group