K R I S T E N T R I P I C C H I O

STUDY OF NITROGEN MUSTARD VESICANT INJURY IN BASAL KERATINOCYTES USING THE

SCRATCH WOUND MODEL

INTRODUCTION: MUSTARDS AS VESICANTS

• Vesicant chemical warfare agent – causes blisters to the eyes, skin, and mucosal membranes • Structure & analogs• Alkylating agent via the carbenium ion6

Sulfur mustard Nitrogen mustard 2-chloroethyl ethyl sulfide (CEES)

INTRODUCTION: MUSTARDS AS VESICANTS

• Blistering occurs in a delayed fashion – apoptotic death6

• Injury occurs because the epidermis basal keratinocytes detach from the basement membrane or dermal layer6

• Delayed reepithelialization2

INTRODUCTION: WOUND HEALING & LAMININ 332

• Basal keratinocytes are anchored to the basement membrane via hemidesmosomes7

• Anchoring molecule - holds cells via integrins to collagen fibers in the BM4,8

• Trimeric protein – previous studies on γ2 chain of the trimer1,2

THE SCRATCH WOUND ASSAY

• “Simple, reproducible assay commonly used to measure basic cell migration parameters such as speed, persistence, and polarity.”3

• Different approaches to analysis: change in length, area, track one particular cell

Make scratch in each well

Seed cells into plate

Take images at regular intervals

DataAnalysis

SCRATCH WOUND: PARAMETERS TESTED

• Initial seeding concentration – % confluency• What seeding number will lead to consistent results and will lead to a

nice monolayer after 24 hrs of growth?• Passage number• Does passage number have an effect on the migration rate of the cells?

• Human variation in seeding & scratching• If the same cells are seeded at the same density, does it matter who

seeds or scratches?

SEEDING CONCENTRATION/CONFLUENCY

• Wells were plated with 1.25x10⁵, 2.5x10⁵, 3.75x10⁵, & 5x10⁵ cells/mL• Wells with 3.75x10⁵ & 2.5x10⁵ saw significantly less variation in migration

rate than the higher seeded 5x10⁵ cells/mL concentration

• Wells plated with 2.5x10 ⁵ cells/mL did not result in a complete monolayer• Therefore, 3.75x10 ⁵ cells/mL was chosen as the ideal seeding conc.

0 1 2 3 4 5 6 7 8 90

0.2

0.4

0.6

0.8

1

Average Area Migrated vs. Time (5x10⁵ cells/mL)

Time (hrs)

Wou

nd C

losu

re (%

)

0 1 2 3 4 5 6 7 8 90

0.2

0.4

0.6

0.8

1

Average Area Migrated vs. Time (3.75x10⁵ cells/mL)

Time (hrs)

Wou

nd C

losu

re (%

)

2.5x10 ⁵ cells/mL 3.75x10 ⁵ cells/mL 0 2 4 6 8 10 12 14 16 18 20

0

0.2

0.4

0.6

0.8

1

Control

A1BA1TA2BA2TA3BA3T

Time (hrs)

Wou

nd C

losu

re (%

)

PASSAGE NUMBER

• Compared PAM212 cells with a passage number of p5 & p23• Similar rate of migration

and amount of variation among samples• Average rates of migration

were similar (5.5 & 6.3%/hr) & not statistically significant when including the std. deviations

0 2 4 6 80

0.2

0.4

0.6

0.8

1

Percent Area Migrated (p5)

A1BA1TA2BA2TA3BA3T

0 2 4 6 80

0.2

0.4

0.6

0.8

1

Percent Area Migrated (p23)

B1BB1TB2BB2TB3BB3T

0 2 4 6 80

0.2

0.4

0.6

0.8

1

f(x) = 0.0551076796727001 x + 0.0105333275054626R² = 0.996377653039439

p5 Avg Migration Rate

0 2 4 6 80

0.2

0.4

0.6

0.8

1

f(x) = 0.0633331377291718 x + 0.00039725679253827R² = 0.997928621592209

p23 Avg Migration Rate

HUMAN VARIATION: SEEDING & SCRATCHING

• Lab tech, Amy, and I each seeded our own 6-well plates • We then scratched half of each plate• No notable difference

0 2 4 6 8 100

0.2

0.4

0.6

0.8

1

Average Migration Rates (Scratch compar-ison)

Amy scratch

KT scratch

Time (hrs)

Wou

nd C

losu

re (%

)

0 2 4 6 8 100

0.2

0.4

0.6

0.8

1

f(x) = 0.017225412124581 x + 0.00639255885576161f(x) = 0.0151315745805029 x + 0.00508862237507296

Average migration rates (Seeding compar-ison)

Amy seeding

Linear (Amy seeding)

Time (hrs)

Wou

nd cl

osur

e (%

)

EXPERIMENTAL SETUP

Control

0.1 µM NM

10 µM NM

1 µM NM

Non-Coated Coated (2 µg/mL laminin 332)

Control

0.1 µM NM

1 µM NM

10 µM NMNitr

ogen

Mus

tard

Dos

e St

udy

HYPOTHESIS: As dose increases, migration will decrease. Coated wells will decrease migration.

RESULTS

Control 0.1 1 100

0.2

0.4

0.6

0.8

1

Average Wound Closure after 9 hrs for coated and non-coated wells

Non-coatedCoated

Dose of NM (µM)

Wou

nd C

losu

re (%

)

• Chose 9-hr time point because this was the longest time point prior to the wounds closing

• Overall, coated wells have slower closure rate

• 10 µM non-coated average changed the trend…

RESULTS

• Compared to the 1 µM data, there was a lot of variation present resulting in a large std deviation• Effect of scratch size• 3.4% deviation vs. 38.5%!

• Redoing the results with the two wells with similar sizes resulted in…

0 2 4 6 8 10 12 14 16 18 200

0.2

0.4

0.6

0.8

1

Noncoated 10 µM NM

0 2 4 6 8 10 12 14 16 18 200

0.2

0.4

0.6

0.8

1

Noncoated 1 µM NM

RESULTS

Control 0.1 1 100

0.2

0.4

0.6

0.8

1

Average Wound Closure after 9 hrs for coated & non-coated wells

NoncoatedCoated

Dose of NM (µM)

Wou

nd C

losu

re (%

)

DISCUSSION/CONCLUSIONS

• Preliminary study• Validated assay – confluency & similar scratch sizes are critical• Increasing dose of NM resulted in increased migration rate –

implications, what does this mean?• The coated wells resulted in a decreased migration rate – this

may better represent how the tissue acts in vivo

FUTURE EXPERIMENTS

• Perform experiment with higher doses of NM• Add different parts of the laminin trimer to the media to see if

they result in faster migration• Coat plate with other EM proteins: fibronectin, collagen, etc. • Develop assay to be high throughput & efficient – 96-well

plates (triplicate resulted in large std devs)

ACKNOWLEDGEMENTS

• I would like to thank Dr. Chang, Amy, and Dr. Gerecke for offering endless guidance over the last few weeks and being quick to answer any questions

• I would also like to thank Dr. Aleksunes, Erin, and Jorge for putting in all of your time and effort to making the SURF program so successful

• Lastly, thanks to the NIH Graduation School of Biomedical Sciences for funding my research experience this summer.

BIBLIOGRAPHY

1. Chang et al. Upregulation of Gamma-2 Laminin 332 in the mouse ear vesicant wound model. J Biochem Molecular Toxicology, 2009, 23, p. 172-184.

2. Chang et al. Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model. Toxicology and Applied Pharmacology, 2013, 268, p. 178-187.

3. Cory, Giles. Scratch-Wound Assay. Chapter 2 in Methods in Molecular Biology, 2011, 769, p. 25-30.

4. Decline, F; Rousselle, P. Keratinocyte migration requires α2β1 integrin-mediated interaction with the laminin 5 γ2 chain. Journal of Cell Science, 2001, 114, p 811-823.

5. Jourdan, MM et al. Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury. Radiation Research, 2011, 176, p. 636-648.

6. Kehe, K; Balszuweit, F; Steinritz, D; Thiermann, H. Molecular toxicology of SM-induced cutaneous inflammation and blistering. Toxicology, 2009, 263, p. 12-19.

7. Kirfel, G; Herzog, V. Migration of epidermal keratinocytes: mechanisms, regulation, and biological significance. Protoplasma, 2004, 223, p 67-78.

8. Nguyen et al. Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion. Current Opinion in Cell Biology, 2000, 12, p. 554-562.

QUESTIONS?