NTU H3 MOLECULAR BIOLOGY

Investigating the effects of

Aspirin on Apolipoprotein A-
I synthesis in Human
Intestinal Cells
Wu Yanlong
1. Introduction

1.1 Literature Review

Overview of lipoprotein metabolism (Robert, 2009):

The main lipids in lipoproteins are free and esterified cholesterol and triglyceride. In
triglyceride metabolism, fats are broken down during digestion, enter intestinal cells, and are
re-packaged in chylomicrons. Chylomicrons, secreted via the lymphatic system into
bloodstream, enter the vena cava and circulate until they interact with lipoprotein lipase
(LPL), releasing free fatty acids, which are taken up by cells that store fats. CM remnants
(CMRs) are taken up by liver cells.

Triglycerides are packaged with cholesterol and are eventually transported in bloodstream in
LDL (low-density lipoprotein). LDL transports cholesterol from the liver to all other parts of
the body. LDL can be taken in by cells via endocytosis. In HDL cholesterol metabolism,
HDL, via APOA-I (A1), mediates reverse cholesterol transport by interacting with receptors
on non-hepatic cells, which are cells that are not from the liver.

Atherosclerosis (Lusis, 2000):

LDL may start depositing along the walls of coronary artery. As they start to become
oxidized, it triggers an inflammatory response which recruits macrophages. The macrophages
attempt to engulf the deposited fats but are overwhelmed by the amount of fats, thereby
forming foam cells. As LDL and foam cells continue to build up, the smooth muscle cells
migrate and form a fibrous cap over the deposition. The smooth muscle cells also deposit
calcium. The fibrous cap may rupture and expose the contents within, leading to thrombosis
(blood clot). This leads to complete blockage of coronary artery which leads to heart attack.

HDL is highly protective against atherosclerosis because it helps to remove cholesterol from
artery wall and peripheral tissues, and it also inhibits oxidation of LDL, which is the trigger
for inflammation.

Aspirin:

Aspirin is an anti-inflammatory drug. Previous research has shown that it helps to stabilize
atherosclerotic plaques by down regulation of MMP-2 and MMP-9 expression (Yiqin, 2009).
Matrix metalloproteinase (MMP)-2 and MMP-9 can degrade the extracellular matrix and lead
to disruption or rupture of plaque.

Aspirin was also shown to increase expression of Scavenger receptor class B type I (SR-BI)
in macrophages, which helps to promote cholesterol efflux, thereby giving it atheroprotective
properties (Tancevski, 2006).

1
1.2 Hypothesis

Aspirin increases Apo-AI synthesis in human intestinal cells.

1.3 Rationale and aim of study

Given that cardiovascular disease is the leading cause of death worldwide, the use of aspirin
as a potential pharmaceutical drug to increase HDL levels among patients has important
implications.

In this study, gene expression and protein expression of Apo-AI is being investigated.

2. Methods

Intestinal cells (Caco-2) were incubated without or with aspirin (5 mM) for a total period of
10 hours, after which total RNA was extracted.

The extracted mRNA was reverse transcribed to cDNA and subsequently subjected to PCR
using specific primers for apoA-I. The DNA was run in agarose gel electrophoresis to
visualize the DNA as bands. Intensity (optical density) of the bands was quantified by
densitometric scanning using the Bio-Rad GS-800 densitometer and Quantity One software;
5 replicates were obtained. This quantifies gene expression.

Protein samples from intestinal cells were incubated with primary antibody followed by
Protein A-agarose beads for protein immunoprecipitation. The precipitated proteins were
visualized as bands via polyacrylamide gel electrophoresis (SDS-PAGE). Similarly, the
Intensity (optical density) of the bands was quantified by densitometric scanning using the
Bio-Rad GS-800 densitometer and Quantity One software; 8 replicates were obtained. This
quantifies protein expression.

Mann-Whitney U test was used to check for significant difference, instead of t-test, because
sample size is small; hence we are unable to assume that data is parametric or follows normal
distribution. A one-tailed test was conducted since the hypothesis is directional.

2
3. Results

70

Intensity (Optical Density)

60
50
40
30
20
10
0
A B

Figure 1. Gene expression of Apo- Figure 2. Gene expression of Apo-AI. Mean

AI. Column 1: DNA ladder. Column optical density of gel bands from 5 replicates.
2: Aspirin absent. Column 3: A: Aspirin absent. B: Aspirin present. Bars
Aspirin present represent standard error of mean (SEM).

As seen in Fig 1, gene expression of Apo-AI is higher in intestinal cells incubated with
aspirin compared to those that are incubated without aspirin. Fig 2 shows that the optical
density of the bands has a significant difference (Mann-Whitney p value of 0.00604, p value
<0.05).

100
Intensity (Optical

80
Density)

60
40
20
0
A B

Figure 3. Protein expression of Apo-AI. Figure 4. Protein expression of Apo-

Colum 1: Protein ladder. Column 2: Aspirin AI. Mean optical density of gel bands
absent. Column 3: Protein samples from from 8 replicates. A: Aspirin absent. B:
cells incubated with aspirin Aspirin present. Bars represent SEM.

As seen in Fig 3, protein expression of Apo-AI is higher in intestinal cells incubated with
aspirin compared to those that are incubated without aspirin. Fig 4 shows that the optical
density of the bands has a significant difference (Mann-Whitney p value of 0.00047, p value
<0.05).

3
4. Discussion

4.1 Results support hypothesis and proposed mechanisms

The results support my hypothesis that aspirin increases Apo-AI synthesis in human intestinal
cells. Aspirin likely increases Apo-AI expression by increasing gene expression only. This
can be seen from the fact that there is a more than 10-fold increase in mRNA in the presence
of aspirin but the increase in protein expression is less than 9 times the original. If aspirin
increased Apo-AI expression by making mRNA more stable or by inducing higher rate of
translation, then the increase in protein expression should be more than 10 times. Therefore,
aspirin increases Apo-AI synthesis at the transcriptional level only, and not at the
translational level.

One possible mechanism by which aspirin increases Apo-AI synthesis may be through NF-
kB transcription factor. Aspirin is able to inhibit NF-kB (Kopp, 1994). Recent studies have
shown how activation of NF-kB leads to inhibition of APOC3 gene promoter (Varvara, 2006).
Therefore, by inhibiting NF-kB, aspirin may potentially lead to increased transcription of
APOC3 gene, since the APOC3 promoter is no longer inhibited. Since APOC3 gene is
closely linked to Apo-A1 gene (Karathanasis, 1985), it is possible that Apo-AI is involved in
a similar process.

Another possible mechanism is via the PPAR-alpha pathway. PPAR-alpha agonism increases
the synthesis of apoA1 (Libby, 2002). In simple terms, activation of PPAR-alpha (a receptor)
by PPAR agonists (ligands that bind to PPAR-alpha), leads to increased transcription of
ApoA1 synthesis. Both Hua (2009) and Yiqin (2009) have suggested that aspirin can
upregulate gene expression of PPAR-alpha. Therefore, it is possible that aspirin has the same
effect as PPAR-alpha agonists, since upregulation of PPAR-alpha gene expression results in
more PPAR-alpha which can then bind to more ligands or agonists.

4.2 Alternative studies that are interesting to note

However interestingly studies by Kagawa (1999) show that Aspirin (5 mM) reduced the
Apo-A1 levels in culture medium of human liver cells and suppressed Apo-A1 mRNA
expression to 73% and 85% of the controls. They quantified Apo-A1 mRNA expression in
the same way by reverse transcription and gel electrophoresis of cDNA followed by
densitometry scanning. The researchers further quantified transcription activity by using
luciferase assay. Even though this study seems to contradict our results here, it is crucial to
note that Kagawa performed his experiment on human liver cells while our experiment
utilizes intestinal cells. Different cells types would have different transcription factors. It is
possible that while aspirin inhibits transcription factors in liver cells, it instead activates
transcription factors in the intestinal cells.

Another important point to note is that while Apo-A1 is an important component of HDL, it
is also used in a LDL-like particle. Lipoprotein(a) is a LDL-like particle in which
apolipoprotein (apo) B-100 is disulfide linked to a single large glycoprotein, Apo-A1
(Utermann, 1989). Epidemiologic studies have revealed that high serum Lp(a) is an

4
independent risk factor for atherosclerotic diseases, including coronary artery disease and
stroke (Bostom 1996, Zenker 1986, Stein 1997). Clinical studies have shown that aspirin
lowered serum Lp(a) concentrations to 80% of the baseline values in patients with high
Lp(a) concentrations (>300 mg/L). The percentage of decrease in serum Lp(a) was larger in
patients with high Lp(a) than in patients with low Lp(a) (<300 mg/L) (Masashi, 2002). This
research paper is done by the same team of researcher as that of Kagawa (1999). It further
shows how aspirin may indeed reduce Apo-AI synthesis instead of increasing it.

The effects of aspirin on Apo-AI synthesis may not be the ultimate factor that influences
occurrence of cardiovascular diseases. Apo-A1 can be used in both HDL and LDL-like
particles. Hence, other factors which directly change the ratio of HDL to LDL particles may
be more important in influencing and changing the likelihood of heart diseases.

4.3 Areas of improvement

One area of improvement is that it would be sensible to normalize the optical density of the
gel. This would bring the OD values into a more reasonable range that makes it easier to
interpret. An example is shown in Kagawas research paper where he normalized OD
readings using G3PDH.

One possible problem with the current methods is that PCR may lead to different
amplification of the cDNA. Hoda (2010) has shown using a mathematical model how
after 25 cycles of PCR, different starting concentrations of DNA leads to different rates of
amplification. In our experiment, we ran PCR for 50 cycles, which may lead to even bigger
discrepancy of results. Therefore, we could limit the PCR to 25 cycles instead. Alternatively,
we would also use real-time PCR which may give more accurate and representative results
for comparison.

Given that there are studies that seem to contradict our findings here, further testing is
required to validate if aspirin does indeed increase Apo-AI synthesis in intestinal cells. The
entire experiment should be repeated to check for reliability of results. Testing can be done
on mice to see if it increases HDL levels. If Apo-A1 synthesis increases, HDL levels should
rise too, ceteris paribus. Ultimately, clinical testing may be used to see if aspirin does indeed
increase HDL levels in blood.

4.4 Conclusion

In conclusion, aspirin increased synthesis of Apo-AI in human intestinal cell by increasing

gene transcription, and therefore, more proteins are produced in translation. Aspirin has the
potential to be used more extensively in treatment of patients with high risk of myocardial
infarction. More research into aspirin and its potential benefits are recommended to clearly
establish aspirins role in protection against heart diseases; potential areas of research include:
mechanism by which aspirin induces increased gene transcription of Apo-AI. Given that
heart diseases are one of the leading causes of death worldwide, it is imperative to find novel
methods that are potentially more efficient and effective for better treatment outcomes.

5
5. Bibliography

Ayako Kagawa. 1999. Aspirin Reduces Apolipoprotein(a) (Apo(a)) Production in Human

Hepatocytes by Suppression of Apo(a) Gene Transcription. The Journal of Biological
Chemistry 274, 34111-34115.

Bostom AG, Cupples A, Jenner JL, Ordovas JM, Seman LJ, Wilson PW, et al. Elevated
plasma lipoprotein(a) and coronary heart disease in men aged 55 years and younger. A
prospective study. JAMA 1996;276:544-548.

Hegele, R. 2009. Plasma lipoproteins: genetic influences and clinical implications. Nat. Rev.
Genetics. 10, 109-121.

Hua Y., et. al. 2009. Aspirin inhibits MMP-2 and MMP-9 expressions and activities through
upregulation of PPARalpha/gamma and TIMP gene expressions in ox-LDL-stimulated
macrophages derived from human monocytes. Pharmacology. 2009;83(1):18-25

Ivan Tancevski, et. al. 2006. Aspirin regulates expression and function of scavenger receptor-
BI in macrophages: studies in primary human macrophages and in mice. The FASEB
Journal vol. 20 no. 9 pages 1328-1335

Karathanasis S. K. Apolipoprotein multigene family: tandem organization of human

apolipoprotein AI, CIII and AIV genes. Proc. Natl. Acad. Sci. U.S.A. 1985;82:6374
6378.

Kopp E., Ghosh S. 1994. Inhibition of NF-kappa B by sodium salicylate and aspirin. Science.
1994 Aug 12;265(5174):956-9.

Lusis, AJ. 2000. Atherosclerosis. Nature 407, 233-241.

Masashi Akaike, Hiroyuki Azuma, Ayako Kagawa, et al. 2002. Effect of Aspirin Treatment
on Serum Concentrations of Lipoprotein(a) in Patients with Atherosclerotic Diseases.
Clinical Chemistry September 2002 vol. 48 no. 9 1454-1459

Peter Libby. 2002. Inflammation in atherosclerosis. Nature 42, 868-874.

Sharifian, Hoda. Errors induced during PCR amplification. Swiss Federal Institute of
Technology, Department of Computer Science (2010). http://dx.doi.org/10.3929/ethz-a-
006088024

Stein JH, Rosenson RS. Lipoprotein Lp(a) excess and coronary heart disease. Arch Intern
Med 1997;157:1170-1176

Utermann G. The mysteries of lipoprotein(a). Science 1989;246:904-910.

Varvara Nikolaidou-Neokosmidou, Vassilis I. Zannis, and Dimitris Kardassis. 2006.

Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor B
pathway. Biochem J. 2006 Sep 15; 398(Pt 3): 439450.

6
Yiqin Y., Meilin X, Jie X, Keping Z. 2009. Aspirin inhibits MMP-2 and MMP-9 expression
and activity through PPARalpha/gamma and TIMP-1-mediated mechanisms in cultured
mouse celiac macrophages. Inflammation 32(4):233-41.

Zenker G, Koltringer P, Bone G, Niederkorn K, Pfeiffer K, Jurgens G. Lipoprotein(a) as a

strong indicator for cerebrovascular disease. Stroke 1986;17:942-945.