Lipoproteins and Atherosclerotic Cardiovascular Diseases

Jingying Wang

University of California, Los Angeles, U.S.A.

Keywords: Lipoprotein, Atherosclerosis, Animal Models, Statistic Models.

Abstract: Metabolisms of triglycerides and cholesterol from both the intestine and the liver are important in humans.

Generally, there are three lipoprotein pathways involving chylomicrons, chylomicron remnants, VLDL, LDL,

and HDL. Started in arterial endothelium, accumulation of LDL and proteoglycans will form oxidized LDL

and lead to arterial macrophages. Then, macrophage foam cells will enhance plaque progression. If the plaque

is unstable, it is very likely to develop plaque rupture and form thrombus. To better understand the mechanism

and find medications, it is important to choose adequate animal models based on lipid metabolism and

characteristics of atherosclerosis. The combination of statistical models in recent studies helps to examine the

effects of two independent factors on one dependent factor.

1 INTRODUCTION

Nowadays, people consume more and more food

containing high fat and cholesterol in developed

countries and the likelihood of getting atherosclerosis

increases. To attenuate or avoid the symptoms of

atherosclerotic cardiovascular diseases, it is

necessary to understand the principles behind these

diseases. In addition, the combination of statistical

models to biological research may differentiate the

influence of two independent factors or two

dependent factors, which provide futural experiments

with possibilities to include more factors in one

research.

2 LIPOPROTEINS, RECEPTORS,

ENZYMES, AND

LIPOPROTEIN PATHWAYS

Lipoproteins are complexes with hydrophobic core,

which is formed by triglyceride and cholesteryl

esters, and hydrophilic phospholipids, free

cholesterol, and apolipoproteins. There are seven

classes of lipoproteins: chylomicrons, chylomicron

remnants, very low density lipoproteins (VLDL),

intermediate density lipoproteins (IDL), low density

https://orcid.org/0000-0002-2023-7801

lipoproteins (LDL), high density lipoproteins (HDL),

and lipoprotein (a) (Lp (a)).

Chylomicrons are triglyceride-rich particles

produced by the intestine. The major structural

protein of chylomicrons is apolipoprotein B-48 (Apo

B-48) that cannot be recognized by LDL receptors.

Chylomicron remnants are smaller particles after

removing triglycerides from chylomicrons by

peripheral tissues. These smaller particles are high in

cholesterol and more pro-atherogenic. VLDL are

triglyceride-rich particles produces by the liver, and

they are smaller than chylomicrons. Compared with

chylomicrons, the major structural protein is Apo B-

100 which is a ligand for LDL receptors. IDL, or

VLDL remnants, is a smaller particle after removing

triglycerides by muscle and adipose tissue. Similar to

chylomicron remnants, IDL is enriched in cholesterol

and pro-atherogenic. LDL is derived from VLDL and

IDL, and it is more enriched in cholesterol. Smaller

LDL is more pro-atherogenic than larger LDL

because it has a decreased affinity with LDL

receptors, which leads to a longer retention time, and

they bind more tightly to proteoglycans and are more

likely to be oxidated, which increases the

consumption by macrophages. (Feingold 2021) HDL

is anti-atherogenic because it acts in reverse

cholesterol transport. The major structural protein of

HDL is Apo A-I. Apo A-I helps interaction between

HDL and ATP-binding cassette transport A1

404

Wang, J.

Lipoproteins and Atherosclerotic Cardiovascular Diseases.

DOI: 10.5220/0011371300003438

In Proceedings of the 1st International Conference on Health Big Data and Intelligent Healthcare (ICHIH 2022), pages 404-409

ISBN: 978-989-758-596-8

 2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reser ved

(ABCA1) and ABCG1 that transport cholesterol from

cells to HDL in reverse cholesterol transport.

(Feingold, 2021) Lp (a) is an LDL particle with

apolipoprotein (a) and it is pro-atherogenic. Another

important apolipoprotein is Apo C that is found in

chylomicrons, VLDL, and HDL. Apo C-II is a co-

factor of lipoprotein lipase (LPL) and hydrolyzes

triglycerides, which means that it is anti-atherogenic.

However, Apo C-III is an inhibitor of LPL and

inhibits triglycerides interact with their receptors.

In addition to ABCA1 and ABCG1, several

receptors and transporters are important in lipid

metabolism. LDL receptors determine the LDL level

in the plasma: the high level of LDL receptors is

corresponding to a low LDL level, while the low level

of LDL receptors is corresponding to a high LDL

level. (Feingold 2021) With the delivery of

cholesterol to cells, the level of HMG CoA reductase

decreases, and the level of acyl-CoA cholesterol acyl

transferase (ACAT) increases. As a result, the level of

LDL receptors, which are controlled by HMG CoA

reductase, decreases. When the cellular cholesterol

level is low, the transcription factor SREBP will be

activated, and LDL receptors will be stimulated to

express. In contrast, when the cellular cholesterol

level is high, the SREBP will be inactive, and the

expression of LDL receptors is low. (Feingold 2021)

Similar to ABCA1, AND ABCG1, class B scavenger

receptor B1 (SR-B1) facilitates selective uptake of

cholesterol esters from HDL particles.

There are several enzymes involved in lipoprotein

metabolism. Lipoprotein lipase (LPL) hydrolyzes

triglycerides in chylomicrons and VLDL and forms

chylomicron remnants and IDL. Apo C-II and Apo A-

V are cofactors of LPL, while Apo C-III and Apo A-

II are inhibitors. Lecithin cholesterol acyltransferase

(LCAT) catalyzes the synthesis of cholesterol esters

and transfers free cholesterol from the surface of the

HDL particle to the core and forms cholesterol esters.

(Feingold 2021) This process reduces the

concentration of cholesterol on the HDL particles and

allows the consumption of more cholesterol from

cells. Cholesteryl ester transfer protein (CETP) helps

transfer cholesterol esters from HDL to VLDL,

chylomicrons, and LDL, and triglycerides from

VLDL and chylomicrons back to HDL. (Feingold

2021)

There are three major lipoprotein pathways:

exogenous lipoprotein pathway, endogenous

lipoprotein pathway, and reverse cholesterol

transport. Exogenous lipoprotein pathway starts in

the intestine: dietary lipids are incorporated into

chylomicrons. Triglycerides in chylomicrons are

hydrolyzed into muscle and adipose tissue into free

fatty acids by LPL, and chylomicrons form

chylomicron remnants. (Feingold 2021) Endogenous

lipoprotein pathway starts in the liver with VLDL.

Triglycerides in VLDL are hydrolyzed into free fatty

acids, and VLDL forms IDL. IDL can be further

metabolized into LDL and can be consumed in tissues

via LDL receptors. (Feingold 2021) Reverse

cholesterol transport begins with nascent HDL in the

liver and intestine. Cells donate cholesterol and

phospholipids via ABCA1 to nascent HDL and form

mature HDL. Additional cholesterol can be

transferred to mature HDL via ABCG1, SR-B1, or

passive diffusion. (Feingold 2021) Facilitated by

CETP, HDL transports cholesterol back to the liver

via SR-B1 or indirectly to VLDL or LDL.

3 ATHEROSCLEROTIC

CARDIOVASCULAR DISEASE

Accumulation of LDL, which is one of the major

culprits of atherosclerotic cardiovascular disease, is

due to interaction between positive-charged amino

acyl residues in Apo B-100 with negative-charged

sulfate and carboxylic acid groups of proteoglycans

in the artery wall. Both changes in the core or on the

surface of LDL may enhance atherosclerosis. For

example, enrichment of cholesterol in LDL or Apo E,

Apo C-III, and serum amyloid A can increase the

binding affinity of LDL and arterial wall

proteoglycans. (Borén 2020) In humans, there is an

inclination to develop atherosclerosis at branches and

bifurcations with laminar blood flow and low or

fluctuating shear stress.

There are four subfractions of LDL: large LDL-I,

LDL-II with intermediate size and density, small

LDL-III, and very small LDL-IV. People with

medium plasma triglyceride levels will release

VLDL1 and VLDL2 that is further metabolized into

LDL-II; people with low plasma triglyceride levels

mainly release smaller VLDL and form

predominantly LDL-I, along with some LDL-II;

people with high plasma triglyceride level release

dense LDL-IV due to high level of VLDL, and they

generally lack lipolysis due to inhibition of

overproduced Apo C-III on LPL. (Borén 2020) Small

dense LDL is more pro-atherogenic than larger LDL

because it enters the artery faster and has a longer

retention time due to impaired binding affinity to

LDL receptors. In addition, small dense LDL is

enriched in Apo C-III and glycated Apo B, and

unsaturated cholesteryl esters are more susceptible to

hydroperoxide. (Borén 2020)

Lipoproteins and Atherosclerotic Cardiovascular Diseases

405

With a longer retention time, LDL particles are

more likely to form oxidized LDL and trigger the

entrance of monocytes into the artery. Monocytes

differentiate in the artery and become macrophages

and intensify oxidized LDL that can be consumed by

scavenger receptors like clusters of differentiation-36

(CD36) and form foam cells. Modified LDL triggers

a series of innate and adaptive immune responses and

leads to inflammation. Defective efferocytosis, which

results in non-resolving inflammation, is due to

signals like CD47 in the artery and will lead to the

accumulation of cell debris. (Borén 2020) Apoptotic

cell will stimulate secondary necrosis that results in

unstable plaque, plaque rupture, and later thrombus

formation. Both plaque rupture and plaque erosion

may lead to thrombus formation. With lipid cores or

thin fibrous cap tissue between the lipid core and

blood that reaches the luminal surface, the blood can

enter and core material may leak out. (Borén 2020)

This process forms plaque rupture that always

accompanying by protruding cholesterol crystals. In

contrast, lesions without lipid cores or thick fibrous

cap will not lead to plaque rupture but instead plaque

erosion, where the plaque is intact but endothelial

cells are deficient.

Recent researches show that a

spotty pattern of calcium deposits is prone to be more

dangerous. An elevated LDL-cholesterol level is one

of the risk factors of calcification. (Borén 2020) In

contrast, HDL-mediated efflux of cholesterol inhibits

calcification. (Borén 2020) While the formation of

atherosclerosis is attributed to the accumulation of

oxidated LDL, the relation between lowering

aggressive LDL and lesion area remains unclear.

Similarly, while HDL features anti-inflammatory and

anti-oxidative functions, its role in attenuating lesion

areas is indistinct but probable.

4 CHOICE OF ANIMAL MODELS

Since it is impossible to track the lengthy

development of atherosclerosis in arteries of humans,

it is necessary to observe that in animal models which

are representative of humans. The murine model is

ideal because of its small size and its relative

homogeneity to humans. For example, both the

mechanism of triglyceride-rich lipoprotein inducing

atherosclerosis and Apo A-I lowering atherosclerosis

can be applied to humans. (Daugherty 2017)

However, while a murine transports cholesterol

primarily in HDL, humans utilize LDL. This

difference in lipoprotein profile protects a murine

from atherosclerosis because there is no binding site

of Lp-PLA2 to LDL. Besides, there is a lower

probability to form oxidated LDL and trigger

atherosclerosis, but a higher probability to form

stable plaques. Furthermore, the much higher level of

LDL receptors in the liver in a murine than in humans

leads to lower LDL levels in a murine and a lower

probability to develop atherosclerosis. There is a

lower level of plasma Apo B on LDL in a murine than

in humans. The chylomicron is from the intestine, and

the VLDL is from the liver in humans. However, Apo

B-48 exists in the VLDL, and Apo B-100 exists in

chylomicrons in a murine. Fed with a high-fat high

cholesterol diet, humans are prone to develop

increased plasma cholesterol and triglycerides, while

a murine may develop increased plasma cholesterol

but lowered triglycerides. In addition, the murine

accumulates lesions primarily in the aortic root, arch,

and other side branches instead of coronary arteries

in humans. Recent studies mainly utilize either Apo

E knock-out mice or LDL receptor knock-out mice.

Although Apo E knock-out mice carry more VLDL,

LDL receptor knock-out mice have higher LDL

particles that are more atherogenic. (Getz, & Reardon

2016) However, Apo E has some athero-protective

functions other than lower plasma lipids like anti-

inflammation and anti-oxidation to lower

atherosclerosis, which generalizes how Apo B-

containing lipoproteins influence atherosclerosis

more difficult. (Getz, & Reardon 2016)

While it is

convenient to study characteristics of atherosclerosis

in a murine, there are limitations when applying the

same mechanism to humans.

Pigs are more relevant to humans than murine.

Like humans, pigs transport cholesterol primarily in

LDL, and pigs also have a binding site of Lp-PLA2

to LDL. Moreover, similar to humans, the LDL

receptors level is low in pigs, and a high-fat high

cholesterol diet can stimulate the increase in both

plasma cholesterol and triglycerides. All these

properties determine the susceptibility of

atherosclerosis and unstable characteristics of

plaques. Pigs are inclined to develop lesions at

branches with low shear stress and laminar blood

flow including coronary, and it is more convenient to

observe changes in arteries because of the huge size

of pigs. (Daugherty 2017) However, since there is no

CETP in pigs, the mechanism of atherosclerosis in

pigs is different from that of humans. In addition, the

huge size of pigs will increase the cost of feeding and

increase the difficulty of experiments.

Recently, scientists find great similarities between

hamsters and humans. Hamsters utilize LDL to

transport cholesterol, and they have CETP, which

means that hamsters share a similar mechanism of

atherosclerosis development. Besides, most Lp-

ICHIH 2022 - International Conference on Health Big Data and Intelligent Healthcare

406

PLA2 in hamsters binds to LDL instead of HDL, and

this increases the susceptibility of atherosclerosis and

forms unstable plaques. All these similarities with

humans, along with the small size of hamsters, ensure

that hamsters are one of the most appropriate animal

models when there is a need to how atherosclerosis

may develop in humans. However, information about

the place of atherosclerosis is still limited, and more

experiments about atherosclerosis in hamsters are

needed.

5 LCAT DEFICIENT HAMSTERS

AND STATISTIC MODELS

In recent scientific studies, it is common to combine

statistical models to analyze research results. In

research conducted by Guo, et al., it is noted that aged

male and female hamsters that are deficient in LCAT

develop atherosclerosis with higher plasma oxidative

lipids but not total cholesterols. (Guo, Liu, Xu, Ma,

Huang, Gao, Wang, Liu, & Xian 2020)

In the study,

Guo, et al. employ a two-way ANOVA test and linear

regression.

In linear regression, there are three important

values: total variability, remaining residue, and the

explained viability due to regression. Total

variability, or 𝑆𝑆



, can be written as Y − Ȳ, which

stands for the difference between the exact value Y

and the mean Ȳ. Remaining residue, or 𝑆𝑆



, can be

written as Y – Ŷ, and it stands for the difference

between the exact value Y and the value on the model

Ŷ. The explained viability due to regression can be

written as Ŷ − Ȳ, and it stands for the difference

between the value on the model Ŷ and the mean Ȳ.

Based on these three values, the coefficient of

determination R square in linear regression is defined

as:

𝑅



=1−









=1−

∑

(









)





∑

(







)





(1)

When 𝑅



is closer to 1, the linear model is more

accurate. In the study of Guo, et al., the linear model

fits well to samples. In the aorta of male

hamsters 𝑅 = 0.804 , and in female hamsters 𝑅 =

0.862 (Figure 1, 2). In the aortic root of male

hamsters, 𝑅 = 0.602 . Based on these data, it is

reliable to conclude that as the plasma

malondialdehyde (MDA) level increases, the lesion

volume, or the severity of atherosclerosis in the aorta

of LCAT deficient hamsters increases.

In the ANOVA test, a statistic F test uses F value

to compare two variances. First, the formula of

regression sum of squares (SSR) is:

𝑆𝑆𝑅 = 𝑛

∑

(𝑥



−𝑥





)





(2)

Where n is the sample size of group j; 𝑥



is the

mean of group j; and 𝑥





is the mean of all

samples.

Then, the formula of the error sum of squares

(SSE) is:

𝑆𝑆𝐸 =

∑∑

(𝑥



−𝑥



)





(3)

Where 𝑥



is the i

term in group j; and 𝑥



 is the

mean of group j.

The total sum of squares (SST) is defined as:

𝑆𝑆𝑇 = 𝑆𝑆𝑅 + 𝑆𝑆𝐸. (4)

Finally, based on formulas (2), (3), and (4), the F

value in F test is:

𝐹=

 

 



 





 















(5)

Where k is the number of groups; and n is the total

sample size.

Based on the calculated F value and the table of

critical values of the F distribution, it is convenient to

find out the P-value that stands for the probability of

coincident results. If the calculated F value is larger

than the critical F value, then it is statistically

significant to reject the null hypothesis that the

variance between the means of two samples has no

significant difference. In the study of Guo et al., P-

values for aorta in male hamsters and aortic root in

female hamsters are less than 0.01, while the P-value

for aortic root in male hamsters is less than 0.05

(Figure 1, 2). In addition, a two-way ANOVA test is

applied to consider the influence of both gender and

age on lesion area in the study of Guo et al. Different

from the one-way ANOVA test, a two-way ANOVA

test considers two conditions: the influence of gender

only on the lesion area and the influence of age only

on the lesion area. Since all P-values for lesion areas

in the aorta and aortic root are less than 0.01 in male

hamsters and 0.001 in female hamsters, it is

statistically significant to conclude that LCAT

deficient hamsters tend to develop higher plasma

MDA levels and larger lesion areas than wild-type

hamsters (Figure 3, 4).

6 CONCLUSIONS

Recent studies develop some generalized ideas about

the characteristics of lipoproteins and their pathways

in transporting cholesterol. Defects in lipoproteins,

lipoprotein receptors, transporters, or enzymes will

Lipoproteins and Atherosclerotic Cardiovascular Diseases

407

lead to retention of LDL in the artery and the

formation of atherosclerosis. However, there are still

confusions about the functions of some

apolipoproteins like Apo C-III and Apo E in the

formations of atherosclerosis. Furthermore, since

current animal models of mice are not appliable to

humans in some cases, futural research about how

these defects express on other animal models like

hamsters may be insightful. When conducting

research, the employment of statistical models helps

to differentiate the influence of various independent

factors that will reduce potential errors when there are

several sets of variables.

7 SUPPLEMENT

Figure 1: The correlation of plasma MDA level and lesion area in aorta and aortic root in wild-type and LCAT deficient male

hamsters (adapted from Guo, et al. 2020).

Figure 2: The correlation of plasma MDA level and lesion area in aorta and aortic root in wild-type and LCAT deficient female

hamsters (adapted from Guo, et al. 2020).

ICHIH 2022 - International Conference on Health Big Data and Intelligent Healthcare

408

Figure 3: Quantification of atherosclerosis lesion area in aortic roots in male hamsters; Quantification of atherosclerosis

plaque area in the aorta in male hamsters; Plasma MDA levels in male hamsters. Scale bar: 1mm. P<0.01 by Two Way

ANOVA/Bonferronis post-test (adapted from Guo, et al. 2020).

Figure 4: Quantification of atherosclerosis lesion area in aortic roots in female hamsters; Quantification of atherosclerosis

plaque area in the aorta in female hamsters; Plasma MDA levels in female hamsters. Scale bar: 1mm. P<0.001 by Two Way

ANOVA/Bonferronis post-test (adapted from Guo, et al. 2020).

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