ApoA-I/A-II-HDL positively associates with apoB-lipoproteins as a potential atherogenic indicator
Kido et al. Lipids in Health and Disease
ApoA-I/A-II-HDL positively associates with apoB-lipoproteins as a potential atherogenic indicator
Toshimi Kido 3
Kazuo Kondo 2
Hideaki Kurata 1
Yoko Fujiwara 3
Takeyoshi Urata 5
Hiroshige Itakura 4
Shinji Yokoyama 0
0 Nutritional Health Science Research Center, Chubu University , Kasugai, Aichi 487-8501 , Japan
1 Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine , Tokyo 105-8461 , Japan
2 Department of Food and Nutritional Science, Toyo University , Itakura-machi, Gunma 374-0193 , Japan
3 Institute for Human Life Innovation, Ochanomizu University , Tokyo 112-8610 , Japan
4 Shinagawa East One Medical Clinic , Tokyo 108-0075 , Japan
5 Department of Diabetes, Metabolism and Endocrinology, Showa University School of Medicine , Tokyo 142-8555 , Japan
Background: We recently reported distinct nature of high-density lipoproteins (HDL) subgroup particles with apolipoprotein (apo) A-I but not apoA-II (LpAI) and HDL having both (LpAI:AII) based on the data from 314 Japanese. While plasma HDL level almost exclusively depends on concentration of LpAI having 3 to 4 apoA-I molecules, LpAI:AII appeared with almost constant concentration regardless of plasma HDL levels having stable structure with two apoA-I and one disulfide-dimeric apoA-II molecules (Sci. Rep. 6; 31,532, 2016). The aim of this study is further characterization of LpAI:AII with respect to its role in atherogenesis. Methods: Association of LpAI, LpAI:AII and other HDL parameters with apoB-lipoprotein parameters was analyzed among the cohort data above. Results: ApoA-I in LpAI negatively correlated with the apoB-lipoprotein parameters such as apoB, triglyceride, nonHDL-cholesterol, and nonHDL-cholesterol + triglyceride, which are apparently reflected in the relations of the total HDL parameters to apoB-lipoproteins. In contrast, apoA-I in LpAI:AII and apoA-II positively correlated to the apoB-lipoprotein parameters even within their small range of variation. These relationships are independent of sex, but may slightly be influenced by the activity-related CETP mutations. Conclusions: The study suggested that LpAI:AII is an atherogenic indicator rather than antiatherogenic. These sub-fractions of HDL are to be evaluated separately for estimating atherogenic risk of the patients.
HDL; apoA-I; apoA-II; apoB; CETP
High-density lipoprotein (HDL) plays a central role in
cholesterol transport from peripheral tissues to the liver
where it is converted to bile acids for enterohepatic
circulation and excretion, as an essential part of its catabolism.
HDL thus appears as an apparent negative risk factor for
atherosclerotic diseases and is believed to act against
atherogenesis. However, many attempts to increase plasma
HDL failed to prevent vascular diseases, in contrast to
their evident decrease by reduction of a positive risk factor
plasma low-density lipoprotein (LDL). Complexity of the
HDL pathway that involves so many steps and factors may
make it difficult to optimize its manipulation for
prevention of atherosclerosis.
HDL is a group of small lipid-protein particles
composed of hydrophobic core of cholesteryl ester (CE) with
a small amount of triglyceride (TG) and of surface layer
of phospholipid, unesterified cholesterol and
apolipoproteins that bind to lipid by amphiphilic helices [
particles are physicochemically unstable and
metabolically active so that their structure is in a dynamic
equilibrium rather than stable construction. HDL is therefore
found with subsets of the particles based on parameters
such as density, size, or apolipoprotein composition.
However, these HDL subgroups have not clearly been
defined for their functions in cholesterol transport or
roles in atherogenesis. We recently reported that
traditional two major subsets of human HDL classified by
apolipoprotein composition, the particles with
apolipoproteins A-I (apoA-I) but not A-II (apoA-II) (LpAI) and
those having both (LpAI:AII), are distinct from each
other with respect to structural stability and apparent
metabolic fate by analyzing 314 plasma samples from a
Japanese cohort study [
]. The former particles are of
variational structure and concentration being composed
of 3 to 4 apoA-I molecules and determine total plasma
HDL level. On the other hand, the latter particles appear
with stable structure containing two apoA-I and one
disulfide-dimeric apoA-II molecules and little-variable
plasma concentration regardless of total HDL level.
Thus, these two HDL subsets seem profoundly different
in structure and metabolism. It is therefore important to
clarify their functional differences to understand
cholesterol transport by HDL which is overall considered as
antiatherogenic, and to design the strategy for
prevention of atherosclerosis by manipulating HDL
metabolism. We here intended to carry out further bioinformatic
analysis of these HDL subsets to examine their differential
roles in atherogenesis.
Fasting blood plasma samples were collected from the
subjects randomly selected from those who visited
Omiya City Clinic for regular health check-up being
blinded for background information, 177 males and 137
females, as reported in detail in the previous
CETP genotype was determined for intron 14
(1452G–A) and exon 15 (D442G) mutations which are
known to explain most of genetic CETP activity
deficiency in Japanese [
]. One male homozygote and 20
male and 17 female heterozygotes were identified among
the subjects above as previously reported [
Total cholesterol (TC), TG, and HDL-cholesterol
(HDL-C) levels in plasma were determined by enzymatic
methods by using commercially available assay kits
(SEIKEN T-CHO(S), SEIKEN FG-TG(II), SEIKEN
HDLCHO, respectively, DENKA SEIKEN, Ltd., Tokyo) in a
biochemical analyzer TBA-60R (TOSHIBA MEDICAL
SYSTEMS CORPORATION, Tochigi, Japan). ApoA-I,
apoA-II and apolipoprotein B (apoB) were determined
by using commercial immunoturbidimetry assay systems
(apoA-I auto 2, apoA-II auto 2, apoB auto 2 Daiichi
Pure Chemicals Co., Ltd., Tokyo) [
]. All procedures of
measurement above mentioned were done automatically
with a biochemical analyzer COBAS MIRA (Roche
Diagnostics Corporation, Indianapolis, USA). LpAI was
evaluated by measuring apoA-I concentration in the
supernatant after immuneprecipitation of LpAI:AII with
anti apoA-II antibody, and LpAI:AII was estimated by
subtracting this value from total apoA-I as well as by
plasma apoA-II concentration [
]. ApoA-I in LpAI was
thus determined as apoA-II-unassociated apoA-I by
using the HYDRAGEL LPAI PARTICLES Kit (Sebia,
Issy-les-Moulineaux, France) by electroimmunodiffusion
] as previously reported [
], being validated
by a method of combination of immuneprecipitation of
LpAI:AII with anti-apoA-II antibody and turbidimetric
immunoassay with anti apoA-I antibody [
Correlation between two sets of the data was analyzed
by simple linear or logarithmic linear regression
performed along with evaluation of significance of
correlation by using GraphPad Prism 5 for Windows
(GraphPad Software, San Diego, CA).
Results and discussion
Of the 314 cohort subjects, one male homozygote of
CETP mutation (D442G) and one CETP normal male
with very high TG (1438 mg/dl) were excluded from the
analysis hereafter. Relationship of HDL apolipoprotein
markers including those for its subfractions, total
apoAI, apoA-I in LpAI and LpAI:AII and apoA-II, to apoB, an
apolipoprotein parameter to indicate particle number of
LDL and very low density lipoprotein (apoB- lipoproteins),
is displayed in Fig. 1. No significant correlation was found
between total apoA-I and apoB. However, apoA-I in LpAI,
measured as apoA-II-unassociated apoA-I and a
determinant of total apoA-I, showed strong inverse relationship
to apoB, as 50% decrease over an increase of apoB by a
factor of 4. In contrast, apoA-II was found increased at
most 25% with significant positive correlation to apoB.
Accordingly, apoA-I in LpAI:AII also showed positive
correlation to apoB to a similar extent. Metabolic nature of
LpAI and LpAI:AII thus appear distinct from each other
demonstrated by their opposite correlation to
Table 1 lists correlation of these HDL subfraction
markers as well as total HDL markers such as HDL-C to
other indicators of apoB-lipoprotein, TG, nonHDL-C
(TC – HDL-C) and TG + nonHDL-C, in addition to
apoB. The relationships of the HDL markers to the
apoB-lipoprotein indicators were found mostly similar to
what were found with those to apoB. Total apoA-I was
however found in significant negative correlation only
with TG and TG + nonHDL-C. On the other hand,
HDL-C was in strong inverse correlation with all of the
apoB-lipoprotein indicators. The findings were
essentially similar among the subgroups of males and
females of the CETP-normal and the CETP mutants,
except that the CETP mutants appeared with reduced
significance (Fig. 1 and Table 2).
The overall results showed a clear difference between
the HDL particles without apoA-II (LpAI) and those
with apoA-II (LpAI:AII) with respect to their relations
to apoB-lipoprotein metabolism. The findings indicate
that LpAI:AII is apparently an atheorgenic index, being
functionally distinct from an apparent antiatherogenic
Classification of HDL into the particles with and
without apoA-II (LpAI:AII and LpAI) is one of the classical
differentiations of human HDL subfractions [
though functional and structural differences of these
particles have not fully and clearly been distinguished.
Nevertheless, LpAI is said to be more responsible for
anti-atherogenic nature of HDL because increase of
HDL is mostly in this subfraction [
] or it seems
biologically more active in such reactions as cell
cholesterol removal , endothelial protection [
selective CE uptake by scavenger receptor BI [
relevance of such hypotheses has, however, not been
fully confirmed [
]. Views and opinions on
metabolic stability of these HDL subfractions are also
Plasma lipoproteins compose the two major
cholesterol transport systems, apoB-lipoproteins for normal
direction from the liver to the somatic cells and HDL for
reverse transport from the peripheral cells to the liver,
which are essentially under independent regulation as to
gene expression related to each pathway. However,
CETP shunts these two pathways by mediating net
transfer of CE generated in HDL to apoB-lipoproteins
creating a by pass for the reverse transport of cholesterol
to the liver via the hepatic LDL receptor. This net move
of CE from HDL to apoB-lipoproteins is generated by
equimolar exchange of CE in HDL and TG in
apoBlipoprotein, so that increase of plasma TG reduces
]. This may cause not only decrease of HDL-C
but also change in other HDL indicators including its
diameter due to subsequent remodeling of the HDL
]. The present data indeed showed strong
inverse relationship of HDL-C to TG (Tables 1 and 2).
We recently reported that distribution of apoA-II
among HDLs to form LpAI:AII HDL is consistent with a
statistical probability model [
] based on the assumption
of higher affinity of apoA-II than apoA-I to lipid particles
]. LpAI:AII particles are structurally stable having
one disulfide-dimer apoA-II and two apoA-I molecules
and its plasma concentration is largely constant regardless
of total HDL concentration . Therefore, variation of
HDL concentration is almost exclusively dependent on
LpAI. The current findings further demonstrated that
inverse relationship to apoB-lipoproteins is found in fact
only with LpAI. In contrast, LpAI:AII positively correlates
with apoB-lipoprotein indicators. We thus conclude that
LpAI and LpAI:AII are metabolically distinct. LpAI:AII is
of stable structure and largely constant concentration, and
shows significant but small increase along with the
increase of apoB-lipoproteins. This relationship may be
modulated by plasma CETP activity as its significance
seems slightly reduced among heterozygous
CETPmutant subjects due to their reduced CE/TG exchange
rate influencing this steady state [
It is clear that apoA-II is the determinant to generate
LpAI:AII. Our knowledge on function of apoA-II is
however very limited. In addition to its higher affinity to
HDL surface [
], apoA-II was shown to enhance
CETP reaction so much as apoA-I [
demonstrated as a much poorer activator of LCAT than apoA-I
]. It is unclear whether these differences interpret
generation of unique LpAI:AII. LpAI:AII may not
directly be resistant to CETP reaction but its structural
stability may act against remodeling caused by CETP
reaction. LpAI, in contrast, seems more variant to
determine total HDL concentration and susceptible to
CETPtriggered remodeling to appear in inverse correlation
with apoB-lipoproteins. Otherwise, the influence by
CETP mutation could merely be caused by relatively
small sample size.
In addition to our previous demonstration that LpAI and
LpAI:AII are distinct from each other structurally and
], we here demonstrated positive
relationship of LpAI:AII with apoB lipoproteins in contrast to the
negative association of LpAI. Accordingly, we predict that
LpAI:AII is an atherogenic parameter in contrast to LpAI
or other HDL parameters as antiatherogenic indicators.
Thus, LpAI and LpAI:AII HDL are functionally different,
with respect to cholesterol transport and accordingly
antiatherogenic activity. This is important information to
understand mechanism of antiathrogeneity of HDL and to
construct strategy for prevention/cure of atherosclerosis
by raising HDL.
apoA-I: Apolipoprotein A-I; apoA-II: Apolipoprotein A-II; apoB: Apolipoprotein
B; CE: Cholesteryl ester; CETP: Cholesteryl ester transfer protein; HDL: High
density lipoprotein; HDL-C: HDL-cholesterol; LDL: Low density lipoprotein;
LpAI: HDL containing apoA-I but no apoA-II; LpAI:AII: HDL containing both
apoA-I and apoA-II; nonHDL-C: TC – HDL-C; TC: Total cholesterol;
The work was supported in part by a grant from Japan Health Sciences
Foundation and by MEXT-supported Program for the Strategic Research
Foundation at Private Universities (S1201007) and by a Grant-in-Aid from
Availability of data and materials
Data sharing is not applicable to this article as no new raw data were
generated in this study.
TK was responsible for collecting and analyzing the data and writing the
manuscript, KK, YF and and HI planned, initiated and monitored the project,
HK analyzed CETP genotypes, TU was involved in validating the methods
and SY was responsible for data analysis and preparing the manuscript. All
the authors approved the final form of the manuscript submitted.
Ethics approval and consent to participate
The protocol for analysis of the blood samples was approved by the Ethics
Committee for Human Gene and Genome Research at Ochanomizu University
]. All the subjects provides with informed consent prior to participation
to the project.
Consent for publication
The informed consent covered agreement for publication of the data.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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