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Localization of the Platelet-Activating Factor Receptor on the Intact

Human Periodontal Ligament and Label-free Imaging of Collagen

Shakeel A. Khan , Michael G. Nichols , Takanari Miyamoto , and D. Roselyn Cerutis
1 2 1 1

Creighton University, School of Dentistry, and 2Creighton University, College of Arts And Sciences.

Abstract Hypothesis
We have reported the presence of the lysophosphatidic acid (LPA) receptor
We hypothesized that a) PDLF on the PDL would express PAF receptors, and that Results B
some of these receptors would be found on and/or around the nuclear membrane,
subtypes (LPARs) LPA1 and LPA3 on the intact human periodontal ligament
where they would likely interact with LPARs (see Fig 2) to regulate the inflammatory This pilot investigation shows that the PAFR can be visualized by
(huPDL) (Cerutis et al. 2016). LPA 18:1 significantly modulates a multitude of
inflammatory transcripts in human gingival fibroblasts (Cerutis et al. 2015)
response to periodontal pathogens; and b) that it would be possible to complement
A) A B C confocal IF in ~ < 0.15 mm-thick pieces of intact human PDL, using
immunofluorescence (IF) detection of PAFR distribution in intact (non-sectioned) 512 x 512 m panoramic of a PDL fragment (20x) an in situ technique we developed (Cerutis et al. 2016). To our
supporting LPARs as having a regulatory role in chronic periodontal disease
formalin-fixed, de-lipidated PDL tissue with SHG visualization of collagen fibril density knowledge, this is the first report of the presence of the PAFR on
(PD). Members of the LPA, platelet-activating factor (PAF) and prostaglandin Representative examples of
(EP) GPCRs can localize to the nuclear membrane in mammals (Gobeil et al.,
and organizational structure. the intact PDL. PDLFs, by virtue of being exposed constantly to
confocal images of the inflammatory bacterial products [including lipopolysaccharide
2003) where they can share signal transduction pathways to activate
inflammatory gene transcription (like COX-2 and inducible nitric oxide R-Phycoerythrin-labeled Ab for (LPS), even in a healthy normal mouth] differ from other fibroblasts
synthase). PAF is found in significantly higher concentrations in saliva and PAFR (pseudo-colored red) and 1 in the body. PDLFs have been shown to possess the capacity to
gingival crevicular fluid from patients with chronic PD (Garito et al. 1995, make a large number of pro- and anti-inflammatory cytokines, and
for DAPI (nuclear stain,
can thus contribute to the progression and eventual outcome of
Keles et al. 2006), as we have reported for LPA (Bathena et al. 2011). Therefore,
if PAFRs are present on the PDL, PAF may act with LPA to regulate periodontal Methods & Materials pseudo-colored blue); shown
as merged signals.
inflammation in periodontal disease (Verardi et al., 2007, Jnsson et
al. 2011).
inflammation in response to periodontal pathogens. Objectives: We All teeth were obtained during routine third molar extractions or periodontal
hypothesized a) that the PAFR would be expressed in the huPDL, and b) that surgeries, and were de-identified (except for age, gender, and periodontal status) Collagen fibrils are known to be dissolved and phagocytized by PDLF
using the second harmonic generation (SHG) technique would enable before use. in periodontal disease. During their interaction with the patients
detection of collagen fibril density and fibril organizational structure of the The teeth were fixed in 10% buffered formalin until analysis, then de-lipidated in 1:1 immune cells, PDLFs modulate the local immune response. This
PDL - and that this technique would complement confocal microscopy methanol-acetone for 15 min, re-hydrated through graded alcohols, and extensively can change the integrity and health of the PDL in several ways:
visualization of PAFR expression. Methods: Polyclonal anti-PAFR antibodies washed in PBS. through affecting the synthesis of inflammatory mediators, their
PAFR and LPA3 receptor rabbit polyclonal antibodies (Cayman Chemical) were receptors and antagonists; altering PDLF proliferation and collagen
(Abs) were covalently tagged with R-Phycoerythrin and used to label PDL from
covalently tagged with R-Phycoerythrin using a Lightning-Link RPE Conjugation synthesis and/or phagocytosis of collagen fibrils; and synthesis of
IRB-approved, consented donors, using an in situ technique we developed and Cleanup kit (Innova Biosciences) and whole-tooth IF was done using an in situ
(Cerutis et al. 2016). Results: Confocal microscopy confirmed both cellular proteolytic enzymes like matrix metalloproteinases (MMPs) along
technique we developed (Cerutis et al. 2016).
and peri-nuclear/nuclear labeling of PAFRs in PDL fibroblasts. SHG analysis Three-color Images of PDL fragments were obtained using a Leica TCS SP8 MP
with their corresponding inhibitors (Jnsson et al. 2011, Takeuchi-
clearly showed the normal collagen structure, abundance, and organization of confocal microscope at the Creighton University Integrated Biological Imaging Igarashi et al. 2016).
the collagen fibrils of the PDL. Conclusions: To the best of our knowledge, Facility (CU-IBIF). First, the nuclear counter stain (DAPI, 405 nm excitation with The ability to use intact PDL to study and monitor inflammatory lipid
this is the first report of the presence of the PAFR on intact huPDL. The detection bandpass of 410-530 nm) and PAFR (RPE, 552 nm excitation with a mediator GPCR expression and distribution represents a useful and
presence of PAFRs on the huPDL suggests that further investigation of the detection bandpass of 570-650 nm) were obtained using single-photon confocal substantially time-saving method over always having to embed and
interplay of the LPAR and PAFR families is needed to determine their role in imaging. Then, an SHG image of the same field of view was obtained using the section the tissue.
both PDL homeostasis and in chronic PD. This study also shows that SHG femtosecond mode-locked pulse train of a Spectra Physics Mai Tai Ti:S laser Our data show that more study is needed to determine what
(Newport Corporation, Irvine, CA, USA) operating at 920 nm with non-descanned constitutes typical PAFR expression in the normal and periodontally
effectively complements confocal microscopy as a tool to help understand
detection by a Super HyD detector with a detection bandpass of 420-500 nm (HQ
diseased PDL.
PDL architecture at the level of receptors and collagen organization. 460/80, Chroma Technology, Bellows Falls, VT, USA).
Fluorescence lifetime measurements performed with a time-correlated single photon
counting module (SPC 830, Becker and Hickl, Berlin Germany) confirmed prompt
1) The nuclear/perinuclear distribution of the PAFR in PDLF strongly
SHG was the most significant emission following 920 nm illumination. supports our hypothesis that this receptor is able to interact with
Periodontal ligament fibroblasts (PDLF) contribute to the biology and stability C LPARs and EP receptors to regulate inflammation in the PDL (as
A postulated; see Fig. 2).
of the attachment apparatus [periodontal ligament, (PDL)] in health and in
periodontal disease. PDLF make the structural extracellular matrix (ECM) 2) SHG effectively revealed the density and 3-D organization of
macromolecules of the PDL like collagen (Howard et al. 1998 and Havemose- PAFR expression in PDL fibroblasts of the intact human collagen fibers in mm-sized, intact human PDL tissue fragments.
Poulsen et al.1997)]. PDLF remodel the attachment apparatus in response to PDL (40x) 3) Being able to visualize the density and organization of the collagen
inflammatory stimuli, which contributes to sustaining inflammation in fibrils provides a much-needed, complementary method to study
periodontal disease (Verardi et al. 2007, Jonsson et al. 2011). Because of this A) Energy level diagram
changes in this important ECM molecule relative to the expression
capacity, the repertoire of inflammation-related receptors expressed by PDLF A) C) illustrating the difference of inflammation-related receptors like the PAFR.
has major relevance for periodontology (Harrel & Nunn 2009) and between conventional single-
photon and simultaneous two-
implantology (El-Mekawy et al. 2013).

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This research was conducted at the Integrative Biological Imaging Facility at Creighton University, Omaha, NE. This facility, supported by the C.U. Medical School, was constructed with support from grants from the National Center for Research
neutrophil responses, including ROS production and exocytosis of Resources (5P20RR016469) and the National Institute for General Medical Science (NIGMS) (8P20GM103427), a component of the National Institutes of Health (NIH). This investigation is solely the responsibility of the authors and does not necessarily
intracellular granules and vesicles (Futosi et al., 2013). represent the official views of NIGMS or NIH.