1. INTRODUCTION
ABSTRACT
References
1
JOHNS HOPKINS UNIVERSITY BLOOMBERG SCHOOL OF PUBLIC HEALTH,
2
JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE
METHODS
Randy Cruz1
, William Aisenberg2
, Niranjana Natarajan2
, Jessie Huang1
, A-Rum Yoon1
, Lakshmi Santhanam2
,
Jennifer Pluznick2
, and Steven S. An1
RGD-coated ferrimagnetic beads (4.5 um in diameter) bound to the
cytoskeleton through cell surface integrin receptors of adherent ASM cells
are magnetized horizontally and then twisted in a vertically aligned
homogenous magnetic field that varied sinusoidally in time. Forced bead
motions are impeded by mechanical stresses developed within the cell
body, and the ratio of specific torque to lateral bead displacements is taken
as a measure of cell stiffness in units of Pa/nm.
RGD-coated ferrimagnetic beads (4.5 um in diameter) bound to the
cytoskeleton through cell surface integrin receptors of adherent ASM cells
are magnetized horizontally and then twisted in a vertically aligned
homogenous magnetic field that varied sinusoidally in time. Forced bead
motions are impeded by mechanical stresses developed within the cell
body, and the ratio of specific torque to lateral bead displacements is taken
as a measure of cell stiffness in units of Pa/nm.
Many sensory receptors have been found in non-sensory
tissues and play important physiological roles. For
example, bitter taste receptors (TAS2Rs) are expressed in
human airway smooth muscle (ASM) cells, and have
been shown to relax ASM and cause bronchodilation1
.
Olfactory receptors (ORs) are G-protein coupled
receptors (GPCRs) and are the largest family of genes in
human genome. They have been found to be expressed in
various tissues and have been shown to play roles in
sperm chemotaxis2
, muscle regeneration3
, and glomerular
filtration rate4
. Expression, signaling and function of ORs
in human ASM cells are not known.
The goal of this project is to identify and characterize
physiologic function of olfactory receptors in human
ASM cells.
SUMMARY & CONCLUSION
A C
5 µm
B D
5 µm
RESULTSRESULTS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 60 120 180 240 300
Time (s)
CellStiffness(ratioofbaseline)
Contraction Response to Histamine
MAGNETIC TWISTING CYTOMETRY (MTC): Dynamic changes in
stiffness were measured as an indicator of the single-cell contraction and
relaxation of isolated human ASM.
PRIMARY HUMAN AIRWAY SMOOTH MUSCLE (ASM)
CELLS: ASM cell cultures were established from lungs unsuitable
for transplantation or from the 4th
and 5th
order bronchi of surgical
lobectomies and pneumonectomies performed for malignancy.
Cells were grown until confluence at 37o
C in humidified air
containing 5% CO2.
METHODS
PCR: RNA was isolated from human ASM cells using a RNeasy kit
(Qiagen) and then treated with DNase. Isolated RNA was subjected to
reverse transcription (RT; SuperScript II, Invitrogen) using degenerate
primers capable of amplifying the entire repertoire of mouse ORs. The
resulting 389 bp product was then TOPO-cloned and sequenced to
identify individual ORs expressed in human ASM cells.
Hwan Mee Yong, Abraham Isak
Acknowledgements
References
RESULTS
A)
Golf AC3B) Figure 1. Human airway smooth
muscle cells express olfactory
receptors and necessary
downstream effectors. A) Five
different ORs are expressed in
human ASM cells. B) OR
downstream effectors are also
expressed.
Figure 2. Addition of SCFAs to human ASM
cells causes cell mechanical changes. A) A
measurement of the baseline cell stiffness after
addition of SCFAs to human ASM cells. The final
concentration of the SCFAs used was 10 mM. B)
ASM cell response to histamine after treatment
with SCFAs. C) Cytoskeletal dynamics after
treatment.• Identify novel sensory olfactory receptors in human
airway smooth muscle cells.
• Characterize physiologic functions of olfactory
signaling in human airway smooth muscle cells.
0 60 120 180 240 300
0
100000
200000
300000
400000
Time (s)
MeanSquareDisplacements
(nm2
)
Untreated
Propionate (0.1mM)
Propionate (1mM)
Propionate (10mM)
A) B)
Figure 3. Dose dependency of human ASM cell cytoskeletal dynamics. Mean square displacement of
human ASM cells after treatment.
0.0
0.2
0.4
0.6
0.8
1.0
FractionProliferating
(EdUpositive/Totalnuclei)
Untreated Formate Acetate Propionate
A) B)
Figure 4. SCFAs have an inhibitory effect on human ASM cell proliferation. A) EdU assay for
proliferating cells. Proliferating nuclei are red and general nuclei are blue (DAPI stain). B) Quantification of
the EdU assay in A) comparing the amount of cells incorporating the modified nucleotide to the total nuclei
present.
0 2 4 6 8
0
20
40
60
Days in Culture (10% FBS)
LiveCellCount(x104
)
Untreated
Formate
Acetate
Propionate
Figure 5. Propionate and Acetate
inhibit cell proliferation of
normal human ASM cells..
0 2 4 6 8
0
20
40
60
80
100
Days in Culture (10% FBS)
LiveCellCount(x104
)
0 2 4 6 8
0
20
40
60
80
100
Days in Culture (10% FBS)
LiveCellCount(x104
)
0 2 4 6 8
0
20
40
60
80
100
Days in Culture (10% FBS)
LiveCellCount(x104
)
Asthma Lung 1
Asthma Lung 2
Asthma Lung 3
Asthma Lung 4
Asthma Lung 5
A) (Untreated) B) (Acetate)
C) (Propionate)
Figure 6. Propionate has a consistent and
inhibitory effect on asthmatic cell proliferation.
A) & B) Show inconsistent proliferation after no
treatment or acetate treatment, respectively,
dependent on cell line. C) Propionate treated cell
lines failed to proliferate.
1.Deshpande, Deepak A., Wayne C H Wang, Elizabeth L. Mcilmoyle, Kathryn S. Robinett, Rachel M. Schillinger, Steven
S. An, James S K Sham, and Stephen B. Liggett. "Bitter Taste Receptors on Airway Smooth Muscle Bronchodilate by
Localized Calcium Signaling and Reverse Obstruction." Nature Medicine 16.11 (2010): 1299-304.
2.Spehr, M. "Identification of a Testicular Odorant Receptor Mediating Human Sperm Chemotaxis." Science 299.5615
(2003): 2054-058.
3.Griffin, Christine A., Kimberly A. Kafadar, and Grace K. Pavlath. "MOR23 Promotes Muscle Regeneration and
Regulates Cell Adhesion and Migration." Developmental Cell 17.5 (2009): 649-61.
4.Pluznick, J. L., D.-J. Zou, X. Zhang, Q. Yan, D. J. Rodriguez-Gil, C. Eisner, E. Wells, C. A. Greer, T. Wang, S. Firestein,
J. Schnermann, and M. J. Caplan. "Functional Expression of the Olfactory Signaling System in the Kidney.” Proceedings of
the National Academy of Sciences 106.6 (2009): 2059-064. Web.
SPONTANEOUS NANOSCALE TRACER MOTIONS: Spontaneous
bead movements allow for the tracking of cytoskeletal remodeling in a
living cell.
CHARACTERIZATION OF BEAD
MOTIONS
MSD(t) ~ D* (t/to)α
The trajectories of bead motions were
characterized by computing the mean
square displacement (MSD) of all beads
as function of time [MSD(t)] (nm2
).
0 60 120 180 240 300
0
100000
200000
300000
Time (s)
MeanSquareDisplacements
(nm2
)
Untreated
Propionate (0.1mM)
Propionate (1mM)
Propionate (10mM)
0 60 120 180 240 300
0
100000
200000
300000
Time (s)
MeanSquareDisplacements
(nm2
)
Untreated
Acetate (0.1mM)
Acetate (1mM)
Acetate (10mM)
C) D)
• Human ASM cells express olfactory receptors and their obligate downstream
effectors.
• Propionate and acetate, the ligands for OR51E2, cause decreases in stiffness
and cytoskeletal remodeling dynamics of human ASM cells.
• Propionate and acetate inhibit human ASM cell proliferation.
Physiological Roles of Olfactory Receptors in Human Airway Smooth Muscle Cells
0 60 120 180 240 300
0
100000
200000
300000
400000
500000
600000
Time (s)
MeanSquareDisplacements
(nm2
)
Untreated
Acetate (0.1mM)
Acetate (1mM)
Acetate (10mM)
Recent discoveries have shown that “sensory” receptors are expressed in non-
sensory tissues and play important physiological functions. Bitter taste receptors
(TAS2Rs) are expressed in the lung where they mediate bronchodilation.
Olfactory receptors (ORs) are expressed in the kidney where they mediate blood
pressure regulation. Using degenerate PCR approach, our labs (Dr. An and Dr.
Pluznick) have now identified 5 ORs that are expressed in human airway smooth
muscle (ASM)–the end effector of acute airway narrowing in asthma. The goal
of my summer project was to characterize physiologic function of one of the
ORs, OR51E2, because the cognate ligands for this receptor have been
previously identified (i.e. short-chain fatty acids (SCFAs)). We used Magnetic
Twisting Cytometry (MTC) to assess the effects of SCFAs on single-cell
contraction and used Spontaneous Nanoscale Tracer Motions (SNTM) to probe
the rate of cytoskeleton remodeling. SCFAs, specifically propionate and acetate,
decreased human ASM cell stiffness and cytoskeletal remodeling dynamics in a
time- and dose-dependent manner. Most strikingly, propionate and acetate
markedly inhibited human ASM cell proliferation as assessed by EdU assay as
well as live cell counting. The inhibitory effects of propionate and acetate on cell
proliferation were universal and were efficacious in human ASM cells isolated
from both normal and asthmatic lung donors. These findings, taken together,
suggest that expression, signaling and function of human ASM ORs could be
exploited for the potential development of new therapies for obstructive lung
disease.
Acetate Propionate
4hr
24
hr
EdU ASSAY: Proliferating human ASM cells take up modified
fluorescent nucleotide during DNA synthesis. The proliferating
cells are quantified using azide and alkyne chemistry.
Human ASM cells express olfactory machinery
Effects of SCFAs on human ASM cell mechanics
Effects of SCFAs on human ASM cell proliferation using EdU Assay
Goals
Effects of SCFAs on human ASM cell proliferation using live cell counts
Expression, signaling and function of human ASM ORs can be further exploited
for the potential development of new therapies for obstructive lung disease.
A) B)
C)
U
ntreated
Form
ate
Acetate
Propionate
0.0
0.1
0.2
0.3
0.4
0.5
BaselineCellStiffness
(Pa/nm)
0 60 120 180 240 300
0.5
1.0
1.5
2.0
Time (s)
CellStiffness
(ratiobaseline)
Untreated
Formate
Acetate
Propionate
0 60 120 180 240 300
0
100000
200000
300000
400000
500000
Time (s)
MeanSquareDisplacements
(nm2
)
Untreated
Formate
Acetate
Propionate
10µM Histamine