Dauer 1

Purpose/Objectives:
Electronic cigarettes (E-cigarettes) have become increasingly popular in the past few years.
These are marketed as a safer alternative to cigarette smoking, as a way to feed nicotine
cravings, and as a recreational activity involving various e-juice flavorings (Rahman,
Mohamed, & Jamshed, 2015). Studies have been done to investigate the risks of E-cigarette
usage based on its different components, but few studies have concluded distinct health risks
related to E-cigarette use. There are also no federal regulations on E-cigarettes in the United
States, which contributes to the lack of safety information on this product (Harrell, Simmons,
Correa, Padhya, & Brandon, 2015). Most of the studies that have found some correlation though
to health issues in humans and E-cigarettes found the e-juice flavorings to be the possible
causative agent. There is unfortunately very little research on the particular effects of propylene
glycol to human cells. I plan to test the effects of propylene glycol (PG) verses E-cigarette
flavorings on vascular smooth muscle cells (VSMCs) through various lab techniques, to
investigate if PG has a detrimental effect on these cells. I plan to look specifically for VSMC
proliferation and cytokine production, which is an effect of inflammation possibly leading to
vascular diseases.

Background:
Proliferation of VSMCs is one of the primary causes of atherosclerosis (arterial wall
inflammatory disease). When a vessel is damaged (through various exogenous or endogenous
stressors such as cigarette smoke or turbulent flow within the vessel) endothelial cell
permeability increases, lipids and macrophages accumulate into the intima of the vessel
containing VSMCs, and macrophages release growth factors which stimulate the VSMCs to

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proliferate to promote vessel healing. The accumulation of VSMCs along with lipids and
inflammatory cells creates plaques leaving to atherosclerosis and further cardiovascular damage
(Ip et al., 1990). During formation of atherosclerosis, increased production of pro-inflammatory
cytokines such as interleukin-1Beta (IL-1B) also affects VSMC proliferation (Libby, Ridker, &
Hansson, 2009). The production of pro-inflammatory cytokines causing VSMC proliferation is
induced through various pathways, some beginning with angiotensin II for example (Han et al.,
2015). COPD is another example of an inflammatory disease related to damage of VSMCs. One
study found that interleukin-6 (IL6), another pro-inflammatory cytokine, could be used to
predict COPD development in cigarette smokers (Emami & Zaerin, 2015). This leads to the role
cigarette smoke in causing vascular inflammation and vascular diseases such as atherosclerosis
and COPD. While cigarettes are known to cause a number of health issues, no primary evidence
has been found for vascular problems related to E-cigarettes. E-cigarette components vary
between different brands, but most contain the basic components of nicotine, PG (used to
produced the inhaled vapor), and various flavorings (Kosmider et al., 2014). PG from Ecigarettes has not been found to cause health risks in humans, but one study found that PG
caused an oxidative stress response and impaired growth in Caenorhabditis elegans, leading me
to wonder how PG would affect other models such as mice or even humans (Panitz, Swamy, &
Nehrke, 2015). Another study found, though, that the flavorings in E-cigarettes caused
cytotoxicity independent of PG (Farsalinos et al., 2013). The model that will be used in this
study is murine VSMCs which are cost-effective and easier to obtain for an undergraduate
research study as opposed to human VSMCs. Limitations for this model will be the time and
materials needed to culture the cells and keep the cells viable which could be affected through
human error (Xu et al., 2009).

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Hypothesis:
Increased concentration of propylene glycol, independent of electronic cigarette flavorings, will
result in increased expression of IL-1Beta and IL6, indicating increased proliferation of murine
vascular smooth muscle cells.

Experimental Design/Budget:
To begin the experiment, murine VSMCs will be cultured using the protocol according to the
ATCC Animal Cell Culture Guide (American Type Culture Collection [ATCC], 2014).
Specific Aim Number 1: Observe proliferation in VSMCs after incubation with various PG
concentrations and E-cigarette flavorings
First, I plan to observe the effects of different concentrations of propylene glycol and different
concentrations of E-cigarette flavorings on mice VSMCs in a dose dependent study. Different
PG dilutions and flavoring dilutions will be prepared and incubated with VSMCs at 80%
confluence. To determine if proliferation has occurred, I will use western blotting to identify
PCNA (proliferating cell nuclear antigen), a marker of cell proliferation (Mitra, Jia, Gangahar,
& Agrawal, 2009). The western blot protocol according to Sigma Aldrich will be used (SigmaAldrich, 2016). This step will require PCNA primary antibody and anti-mouse secondary
antibody. If PCNA is found using western blotting, I will take the concentration of PG that
caused the most production of PCNA and use this concentration in a time course study. The
same will be done for the cells incubated with the flavorings. VSCMs will be incubated with
this concentration of PG (or flavorings) for 0, 15, 30, 60, 120, and 1440 minutes. The same
western blotting technique used above will be used to determine proliferation in the VSCMs.

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The time course study will determine the amount of time for PG (or flavorings) to cause
proliferation, which will give an idea as to length of time of e-cigarette usage to damage
VSMCs. If no proliferation is found, I will continue to investigate specific aim number 2 as
there is a possibility of error occurring in the experiments done for specific aim number 1. If
proliferation is found, I will use the next experiments under specific aim 2 to validate my results
with further, more specific testing.
Specific Aim Number 2: Investigate the role of pro-inflammatory cytokines (IL1B and IL6) in
the proliferation of VSMCs in PG or E-cigarette flavorings
As a positive control, I will stimulate VSCMs with angiotensin II and look for the mRNA of
both IL1B and IL6 using the protocol for RT-PCR according to Thermo Fisher Scientific
(Thermo Fisher Scientific, 2016). The DNA from these cells will need to be isolated and placed
in the PCR machine along with the sequence specific primers for IL1B and IL6. As a negative
control, I will stimulate the VSCMs with saline, and as the first experimental control I will
stimulate the VSMCs with the concentration of PG found to cause proliferation (if no
proliferation was found, I will use an average concentration of PG of the concentrations tested
under specific aim number 1.) For the second experimental control I will stimulate VSMCs with
the concentration of flavorings found to cause proliferation (or an average of the concentrations
tested under specific aim number 1). Both the negative control and experimental controls will be
tested using the same RT-PCR method stated above.
Budget:

Reagent
Propylene Glycol
PCNA primary antibody
Anti-mouse secondary antibody

Cost
50.00 (Sigma-Aldrich)
279.00 (Santa Cruz Biotechnology)
22.00 (Santa Cruz Biotechnology)

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Angiotensin II
Forward and reverse primers for IL1B
Forward and reverse primers for IL6

33.20 (Sigma-Aldrich)
50.00
50.00

Potential Results:
I predict that I will find VSCMs proliferate at at least 100% PG independent of the flavoring. If
this is true, the PCNA protein will be found in the cells treated with this concentration using
western blotting. If no PCNA is found in any of the cell cultures treated with PG, this either
indicates that PG does not cause proliferation or that human error occurred during the
experiment (the western blotting protocol was not performed correctly). If PG does cause
proliferation, then the cells incubated with the flavorings will also proliferate, as the flavorings
are sold as a solution containing PG.
I predict that at least 30 minutes of incubation with PG will cause VSMC proliferation. If this is
true, the PCNA protein will be found in the cells treated with PG for this amount of time using
western blotting. It is possible that PG will proliferate at a certain incubation time, but any extra
time after this the cells die. This could correlate to proliferation eventually causing VSMC
death, or PG activating other cellular pathways leading to cell death. It is also possible that none
of the incubation times used lead to PCNA production, which could either mean that PG does
not cause proliferation, or that human error occurred during the experiment.
I predict that after stimulating the VSMCs with angiotensin II, a high amount of mRNA for
IL1B and IL6 will be found using RT-PCR because angiotensin is known to cause a proinflammatory response. If the mRNA is not found for these cytokines, then human error
possibly occurred (cells were not cultured properly or PCR protocol not done correctly). I
predict that stimulating the VSMCs with saline will lead to no mRNA production of the IL1B
and IL6, because this neutral solution should not cause inflammation (if inflammation is found,

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this could mean the sample was contaminated). I predict that stimulating the VSMCs with PG
will lead to a high amount of mRNA production for IL1B and IL6, which could correlate with
PG causing cell proliferation. If the mRNA is not found, this could either mean that PG does not
cause proliferation or that human error occurred. I also predict that stimulating the VSMCs with
the flavorings will lead to a high amount of mRNA production for IL1B and IL6, because the
flavorings contain PG. If only the flavorings are found to cause proliferation in each
experiment, this would suggest that the flavorings cause proliferation independent of PG.
Impact:
If PCNA and/or pro-inflammatory cytokines are found in VSMCs after proliferation with PG
independent of flavorings, these results suggest that PG causes VSMC proliferation. If PG
causes these cells to proliferate, this suggests that PG and e-cigarette usage could be an
underlying cause to various vascular diseases such as atherosclerosis and hypertension. If these
results are found, this does not prove PG causes vascular diseases, but will possibly recruit other
students to test the effects of PG on living cells. Other students will be able to build on this
study and investigate other pathways to cell proliferation, as it is a complex subject stimulated
by various factors.

References:
Rahman, A., Mohamed, M., Jamshed, S. (2015). Effectiveness of electronic cigarettes: a narrative
review. International Medical Journal, 22(3), 122-131.
Harrell, P., Simmons, V., Correa, J., Padhya, T., Brandon, T. (2015). Electronic Nicotine Delivery
Systems (E-Cigarettes): Review of Safety and Smoking Cessation Efficacy. Otolaryngology
Head Neck Surgery, 151(3), 381-393. doi: 10.1177/0194599814536847.

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Ip, J.H., Fuster, V., Badimon, L., Badimon, J., Tauban, M. B., Chesebro, J. H. (1990). Syndromes of
accelerated atherosclerosis: Role of vascular injury and smooth muscle cell proliferation.
Journal of the American College of Cardiology, 15(7), 1667-1687. doi:10.1016/07351097(90)92845-S
Libby, P., Ridker, P. M., Hansson, G. K. (2009). Inflammation in atherosclerosis: from pathophysiology
to practice. Journal of the American College of Cardiology, 54(23), 2129-38.
doi:10.1016/j.jacc2009.09.009
Han, S., Kim, C., Lee, Y., Kim, D., Han, K., Cha, D. (2015). Pentoxifylline inhibits angiotensin IIinduced proliferation in rat vascular smooth muscle cells. Biomedical Research, 26(4), 744-749.
Emami, A., Zaerin, O., (2015). Role of serum interleukin 6, albumin and c-reactive protein in COPD
patients. Tanaffos Journal, 14(2), 134-40.
Kosmider, L., Sobczak, A., Fik, M., Knysak, J., Zaciera, M., Kurek, J., & Goniewicz, M.L. (2014).
Carbonyl compounds in electronic cigarette vapors: Effects of nicotine solvent and battery
output voltage. Oxford Journals: Nicotine & Tobacco Research, 16 (10). doi:
10.1093/ntr/ntu078
Panitz, D., Swamy, H., & Nehrke, K. (2015). A C. elegans model of electronic cigarette use:
Physiological effects of e-liquids in nematodes. BMC Pharmacology & Toxicology, 16 (32).
doi:10.1186/s40360-015-0030-0
Farsalinos, K., Romagna, G., Allifranchini, E., Ripamonti, E., Bocchietto, E., Todescho, S., Tsiapras,
D., Kyrzopoulos, S., Voudris, V. (2013). Comparison of the Cytotoxic Potential of Cigarette
Smoke and Electronic Cigarette Vapour Extract on Cultured Myocardial Cells. International
Journal of Environmemental Research and Public Health, 10(10), 5146-5162.
doi:10.3390/ijerph10105146.
Xu, S., Chen, J., Xiao, P., Lan, T., Le, K., Cheng, F., He, L., Shen, X., Huang, H., Liu, P. (2009).
Development of an optimized protocol for primary culture of smooth muscle cells from rat
thoracic aortas. Cytotechnology, 61(1-2), 65-72. doi:10.1007/s10616-009-9236-6.
Mitra, A. K., Jia, G., Gangahar, D. M., & Agrawal, D. K. (2009). Temporal PTEN inactivation causes
proliferation of saphenous vein smooth muscle cells of human CABG conduits. Journal of
Cellular and Molecular Medicine, 13(1), 177187. doi:10.1111/j.1582-4934.2008.00311.x.
(2014). ATCC animal cell culture guide: Tips and techniques for continuous cell lines. Retrieved from
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_Guide.ashx

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(2016). Sigma-aldrich western blotting (immunoblotting) protocol. Retrieved from
http://www.sigmaaldrich.com/technical-documents/protocols/biology/westernblotting.html#procedure
(2016). Thermo fisher scientific: PCR & RT-PCR overview. Retrieved from
https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-amplification-andexpression-profiling/pcr-protocol/pcr-and-rt-pcr.html