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Tagging SNP haplotype analysis of the secretory PLA2-V gene, PLA2G5, shows strong association with LDL and oxLDL levels, suggesting functional distinction from sPLA2-IIA: results from the UDACS study


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Animal and human studies suggest that both secretory PLA2 (sPLA2)-V and sPLA2-IIA (encoded, respectively, by the neighbouring PLA2G5 and PLA2G2A genes) contribute to atherogenesis. Elevated plasma sPLA2-IIA predicts coronary heart disease (CHD) risk, but no mass assay for sPLA2-V is available. We previously reported that tagging single nucleotide polymorphism (tSNP) haplotypes of PLA2G2A are strongly associated with sPLA2-IIA mass, but not lipid levels. Here, we use tSNPs of the sPLA2-V gene to investigate the association of PLA2G5 with CHD risk markers. Seven PLA2G5 tSNPs genotypes, explaining >92% of the locus genetic variability, were determined in 519 patients with Type II diabetes (in whom PLA2G2A tSNP data was available), and defined seven common haplotypes (frequencies >5%). PLA2G5 and PLA2G2A tSNPs showed linkage disequilibrium (LD). Compared to the common PLA2G5 haplotype, H1 (frequency 34.9%), haplotypes H2-7 were associated with overall higher plasma LDL (P < 0.00004) and total cholesterol (P < 0.00003) levels yet lower oxLDL/LDL (P = 0.006) and sPLA2-IIA mass (P = 0.04), probably reflecting LD with PLA2G2A. Intronic tSNP (rs11573248), unlikely itself to be functional, distinguished H1 from LDL-raising haplotypes and may mark a functional site. In conclusion, PLA2G5 tSNP haplotypes demonstrate an association with total and LDL cholesterol and oxLDL/LDL, not seen with PLA2G2A, thus confirming distinct functional roles for these two sPLA2s.
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Tagging SNP haplotype analysis of the secretory
PLA2-V gene, PLA2G5, shows strong association
with LDL and oxLDL levels, suggesting
functional distinction from sPLA2-IIA:
results from the UDACS study
Peter T.E. Wootton
, Nupur L. Arora
, Fotios Drenos
, Simon R. Thompson
Jackie A. Cooper
, Jeffrey W. Stephens
, Steven J. Hurel
, Eva Hurt-Camejo
Olov Wiklund
, Steve E. Humphries
and Philippa J. Talmud
Division of Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School,
5 University Street, London WC1E 6JF, UK,
The Medical School, University of Wales Swansea, Singleton Park,
Swansea SA2 8PP, UK,
Department of Diabetes and Endocrinology, UCL Hospitals, London W1T 3AA, UK,
AstraZeneca, R&D, Molecular Pharmacology, Mo
lndal S-43183, Sweden and
Wallenberg Laboratory for
Cardiovascular Research, l, Goteborg SE-413 45, Sweden
Received January 25, 2007; Revised March 19, 2007; Accepted April 6, 2007
Animal and human st udies suggest that both secretory PLA2 (sPLA2)-V and sPLA2-IIA (encoded, respect-
ively, by the neighbouring PLA2G5 and PLA2G2A genes) contribute to atherogenesis. Elevated plasma
sPLA2-IIA predicts coronary heart disease (CHD) risk, but no mass assay for sPLA2-V is available. We pre-
viously reported that tagging single nucleotide polymorphism (tSNP) haplotypes of PLA2G2A are strongly
associated with sPLA2-IIA mass, but not lipid levels. Here, we use tSNPs of the sPLA2-V gene to investigate
the associ ation of PLA2G5 wi th CHD risk markers. Seven PLA2G5 tSNPs genotypes, explaining >92% of the
locus genetic variability, were determined in 519 patients with Type II diabetes (in whom PLA2G2A tSNP data
was available), and defined seven common haplotypes (frequencies >5%). PLA2G5 an d PL A2G2A tS NP s
showed linkage disequilibrium (LD). Compared to the common PLA2G5 haplotype, H1 (frequency 34.9%),
haplotypes H27 were associated with overall higher plasma LDL (P < 0.00004) and total cho lesterol
(P < 0.00003) levels yet lower oxLDL/LDL (P 5 0.006) and sPLA2-IIA mass (P 5 0.0 4), probably reflecting LD
with PLA2G2A. Intronic tSN P (rs11573248), unlike ly itself to be func tional, distinguished H1 from
LDL-raising haplotypes and may mark a functional site. In conclusion, PLA2G5 tSNP haplotypes demonstrate
an association with total and LDL ch olesterol and oxLDL/LDL, not seen with PLA2G2A, thus confirming
distinct functional roles for these two sPLA2s.
Secretory phospholipase A2 (PLA2) group V (sPLA2-V)
enzyme is a member of the superfamily of PLA2 enzymes
characterized by their ability to hydrolyse the sn-2 ester
bond of phospholipids and cell membranes, generating
non-esterified free fatty acids (NEFAs) and lyso-phospholipids
(1). The sPLA2-V gene (PLA2G5) is tightly linked and in a
negative orientation to the sPLA2-IIA gene (PLA2G2A)on
chromosome 1p3436.1 (2), with both enzymes sharing
structural and functi onal similarities (3). While the role of
sPLA2-IIA in atherogenesis has been well studied, the
# The Author 2007. Published by Oxford University Press. All rights reserved.
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Human Molecular Genetics, 2007, Vol. 16, No. 12 14371444
by guest on May 29, 2013 from
involvement of sPLA2-V is less well understood and questions
about functional redundancy have been raised (4).
sPLA2-IIA is an acute phase protein, elevated in cell types in
response to pro-inflammatory stimuli (3). sPLA2-IIA hydrolyses
LDL phospholipids generating oxidation-susceptible, small-
dense LDL (sdLDL) particles, with an altered configuration of
apolipoprotein B (5), leading to LDL-receptor-independent
uptake into the arterial wall (6). Within the arterial wall, these
modified LDL particles bind proteoglycans present in the
intima and are further modified, leading to increased retention
(7). In addition, the release by sPLA2s of potent lipid
mediators, in particular arachidonic acid and lyso-phospholipids,
promotes pro-inflammatory responses in the arterial wall
(reviewed in 8). The involvement of sPLA2-IIA in athero-
genesis is confirmed by its localization in atherosclerotic
plaques (9,10).
Observational studies have also identified sPLA2-IIA as a
marker for coronary heart disease (CHD) risk (11), with elev-
ated levels associated with an increased probability of recur-
rent coronary events (12,13). In prospective analysis, serum
levels of sPLA2-IIA were associated with a higher risk of
future CHD in healthy individuals independent of classical
and inflammatory markers (14). In animal models,
sPLA2-IIA transgenic mice show an increased susceptibility
to atherosclerosis (15,16).
Less information exists regarding the potential
pro-atherogenic actions of the functionally similar sPLA2-V
enzyme. In vitro, sPLA2-V is able to hydrolyse HDL and
LDL phosphatidylcholine (PC) 20 times more efficiently
than sPLA2-IIA (17), thus hydrolysing the most common
phospholipid species present in lipoproteins and cell surface
membranes (18), leading to arachidonic acid and lyso-PC
generation (19). Since several atherogenic susceptible mouse
strains have a naturally occurring mutation in pla2g2a, other
sPLA2s have been implicated in the development of athero-
sclerosis in these strai ns. sPLA2-V is a likely candidate, and
recent immunohistochemical analysis (supported by mRNA
expression studies) has shown sPLA2-V to be associated
with smooth muscle cells and foam cells in the lipid cores
of both human and mouse athe rosclerotic lesions (20). LDL
receptor knock-out mice, either over-expressing or deficient
in Pla2g5, provide the first in vivo evidence that sPLA2-V
contributes to atherosclerosis (21).
Although these studies support an independent role for
sPLA2-V in atherogenesis, currently no commercially avail-
able assay for sPLA2-V mass exists, and therefore human
observational studies to confirm the CHD risk association
cannot be undertaken.
By utilizing a tagging single nucleotide polymorphism
(tSNP) haplotype approach, we have made use of common
genetic variations to test the hypothesis that variation in
PLA2G5 influences both plasma lipid levels and oxidative
stress. These tSNPs were genotyped in a cohort of patients
with Type II diabetes (T2D) mellitu s, a group associated
with a higher CHD risk, where oxidative stress and
lipid measurements were available (22). In addition, we
have sPLA2-IIA mass measures and PLA2G2A tSNP haplo-
type data in the same cohort (22), thus enabling us to
compare the relationship of these two genes with CHD
risk markers.
The baseline characterist ics of the Caucasian men and women
with T2D, with or without CHD, are shown in Table 1. Those
who had CHD were significantly older, and had a longer dur-
ation of diabetes, and their higher usage of statins and angio-
tensin converting enzyme (ACE) inhibitors, most likely
explains their lower diastolic blood pressure (BP), LDL and
total cholesterol levels compared with CHD-free men and
Seven tSNPs (21437C. A, 21251G. A, 2423G. A,
1640C. T, 1742Gin/del, 11069TAin/del, 22507T. G) were
identified in the PLA2G5 gene region. The genotype distri-
bution of all tSNPs was as expected for Hardy Weinberg
equilibrium as tested by THESIAS (Table 2 gives a summary
of the observed allele frequencies). The locations of the
chosen tSNPs (three in the promoter region, three within
introns and one variant 3
of exon 5) are shown schematically
in Figur e 1A. Lewontin
for each individual tSNP is shown
below the SNP map in Figure 1B, demonstrating strong LD
across the PLA2G5 gene.
Univariate analyses of tSNPs with total, LDL- and
HDL-cholesterol and measures of total antioxidant status,
LDL size and oxidized LDL to LDL ratio are presented in
Supplementary Material, Tables S2S8. Of the tSNPs,
21437C.A, 1742Gin/del and 11069TAin/del showed signifi-
cant association with cholesterol and LDL levels (P-values
ranged from P , 0.012,0.0001); Supplementary Material,
Tables S2, S6 and S7, respectively). 2423G.A and
1742Gin/del were also strongly associated with differences
in sPLA2-IIA levels (P , 0.01; Supplementary Material,
Tables S4 and S6, respectively).
Table 1. Baseline characteristics of Caucasian patients with Type II diabetes
from UDACS
No CHD, N ¼ 383 CHD, N ¼ 136 P-value
Age (years) 65.5 (11.3) 69.5 (9.7) 0.0003
BMI (kg/m
29.2 (5.5) 29.5 (4.7) 0.67
HbA1c (%)
7.7 (1.7) 7.5 (1.5) 0.27
Glucose (mmol/l)
10.02 (4.40) 9.58 (4.25) 0.31
Cholesterol (mmol/l) 5.19 (1.07) 4.71 (1.12) ,0.0001
LDL (mmol/l)
2.81 (0.93) 2.32 (0.89) ,0.0001
HDL (mmol/l)
1.30 (0.38) 1.23 (0.37) 0.06
TG (mmol/l)
1.90 (1.06) 1.92 (1.07) 0.84
SBP (mmHg)
141.5 (20.6) 140.0 (20.9) 0.47
DBP (mmHg) 81.2 (11.4) 78.4 (10.0) 0.01
Duration of diabetes
8.0 (416) 11.0 (617) 0.005
Gender (% male) 57.2% (219) 66.2% (90) 0.07
Smoking (% current) 17.0% (64) 12.0% (16) 0.18
TAOS (%)
44.9 (36.752.5) 42.9 (34.150.7) 0.13
Ox-LDL/LDL (U/mmol)
16.8 (7.8) 18.6 (10.3) 0.08
sd-LDL (%)
71.9 (58.581.4) 71.5 (54.980) 0.74
CRP (mg/L)
1.66 (1.42) 1.77 (1.59) 0.49
sPLA2-IIA (ng/ml)
3.08 (2.20) 3.45 (2.62) 0.12
Statin (%) 23.0 60.0 ,0.0001
ACE I (%) 26.5 38.9 0.003
Square-root transformed.
Median (IQR).
1438 Human Molecular Gen etics, 2007, Vol. 16, No. 12
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Figure 1B shows the D
for PLA2 G2A tSNPs used pre-
viously (22), indicating that despite the 90 Kb distance, there
is a certain degree of LD across the region, with 2423G.A
and the 1742Gin/del, PLA2G5 tSNPs that showed significant
association with sPLA2-IIA mass, showing LD with
PLA2G2A tSNPs 2655T.C and the 763C.G, which had
shown strong association with differences in sPLA2-IIA
mass (22).
Since the seven PLA2G5 tSNPs were primarily identified
for tagging purposes, chosen to cover maximum genetic varia-
bility of PLA2G5, haplotype analysis was examined. Complete
haplotype analysis was available on 351 individ uals. Of the
potential 119 haplotypes defined by the tSNPs, 18 inferred
haplotypes were observed in the sample. Of these, seven
occurred at frequencies .5% and accounted for 92% of the
haplotypic variation within the gene (Table 3). Table 4
shows the association of these haplotypes with intermediate
phenotypes, giving the mean value for one copy of each hap-
lotype as determined by THESIAS (23,24). Compared to the
common haplotype, H1, haplotypes H24 were associa ted
with significantly higher cholesterol (9.1%, P ¼ 0.008; 13%,
P ¼ 0.001; 14.0%, P , 0.001, respectively) and H2 was
associated with higher LDL (23%; P , 0.001) and lower
sPLA2-IIA mass (49%; P ¼ 0.001). H6 was associated with
47% (P ¼ 0.01) lower oxLDL/LDL than H1. This effect on
cholesterol and LDL could not be accounted for by statin
usage, as there was no difference in statin used either by indi-
vidual genotypes or haplotypes (all .0.4).
Since the effects of H2 7 showed no significant evidence
for heterogeneity on any trait, their combined effects, com-
pared to H1, were examined. The unadjusted P-values of
these combined haplotypes are presented in Table 4. Com-
pared to H1, H2 7 carriers had significantly lower
sPLA2-IIA mass (P ¼ 0.04), and higher cholesterol
(P , 0.00003) and LDL (P , 0.00004) and lower oxLDL/
LDL (P ¼ 0.006). These P-values remained statistically sig-
nificant even after adjustment for age, gender, CHD status
and statin use (Table 4).
The mean LDL- cholesterol levels in subjects with 0 (H1/
H1), 1 (H1/H27) and 2 (H2 7/H27) copies of the com-
bined H2 7 haplotypes are presented in Figure 2, showing a
strong co-dominant raising effect.
The UDAC study is cross-sectional in design and did not
have the power to assess the association of these haplotypes
with CHD risk since the number of CHD positive individuals
in each haplotype group was small. Attempts at cladogram
analysis similar to that demonstrated in the haplotype analysis
of the closely related PLA2G2A gene (22) was not achievable
since several different combinational changes of tSNPs were
possible for each haplotype. As such, no individual tSNP
could be identified as being responsible for the associations
described. However, H2 7, which showed significant associ-
ation with LDL cholesterol, in contrast to H1, have in
common the 11609TAin allele, while H1 is defined by the
rare 11609TAdel. H5, which also carrie s the 11609TAdel
e, has trait values most similar to H1. The 11609TAin/
del variant (rs11573248) is located 3 kb 5
of exon 2, and it
is therefore doubtful that it is of functional importance, but
is likely to be in strong LD with a yet undetermined functional
PLA2G5 tSNP haplotypes are associated with differences
in LDL levels and sPLA2-IIA mass
We have used a genetic approach to distinguish the effects of
the sPLA2 enzymes group V and IIA. tSNPs that capture 92%
of the locus genetic variation were used to investigate the
association of PLA2G5 variants (encoding sPLA2-V) with
plasma lipid levels and with markers of oxidative stress, con-
trasted in the same study to those previously reported for
PLA2G2A (encoding sPLA2-IIA) (22). The common
PLA2G5 tSNP haplotype H1 (with a frequency of 39%),
when compared with the rarer haplotypes H2 7, showed
strong association with lower plasma levels of LDL
(P , 0.00004) and total cholesterol (P , 0.00003) , yet
higher oxLDL/LDL (P ¼ 0.006). In this same sample, we pre-
viously showed that tSNP haplotypes of PLA2G2A were
strongly associated with sPLA2-IIA mass levels (P ,
0.00001), yet showed no association with lipid levels or
measures of oxidative stress (22). These data implied that in
vivo, sPLA2-IIA is not having a major impact on determining
the hydrolysis of lipids in the circulation. In contrast, PLA2G5
tSNP haplotypes are having a major effect on LDL levels,
reflected also in levels of total cholesterol, and oxLDL levels.
Functional differences in sPLA2-V and sPLA2-IIA
The contrasting effects of PLA2G5 and PLA2G2A on LDL
levels are supported by in vitro data demonstrating that
sPLA2-V is 20 times more active than sPLA2-IIA in its
ability to hydrolyse phospholipids (17). The recent studies
by Rosengren et al. (20,25) strongly support distinct roles
for sPLA2-V and sPLA2-IIA on circulating lipids and in
atherogenic lesions. Recombinant sPLA2-V but not sPLA2
IIA hydrolysed lipoprotein phospholipids in human sera and
isolated VLDL, HDL and LDL (preferentially in that order,
probably due to differences in the sphingomyelin content),
accompanied by an increase in lyso-phospholipids (20). This
enzymatic difference between the two sPLA2s is suggested
to be due to the tryptophan residues in the interfacial-binding
region of sPLA2-V, absent in sPLA2-IIA, which would enable
enhanced penetration of sPLA2 -V into the phospholipid
monolayer (20).
Table 2. SNP rs number and minor allele frequency of the PLA2G5 tSNPs in
tSNP rs number Minor allele frequency
(95% CI)
2 1437C. A rs11573185 0.45 (0.430.48)
2 1251G. A rs2148911 0.07 (0.05 0.08)
2 423G. A rs11573191 0.18 (0.160.19)
1640C. T rs640022 0.15 (0.13 0.16)
1742Gin/del rs11573203 0.28 (0.260.30)
11069TAin/del rs11573248 0.36 (0.340.39)
22507T. G rs622450 0.14 (0.120.15)
Human Molecular Genetics, 2007, Vol. 16, No. 12 1439
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Further differences between sPLA2-V and sPLA2-IIA were
seen by Rosengren et al. (20). In studies of C57BL/6 mice
and ldlr/apoe double knockout mice, which lack pla2g2a due
to deletion of exon 3, sPLA2-V expression was upregulated
in the aorta when mice were fed a Western diet. No effect of
a high fat diet was seen on sPLA2-II A expression in the
PLA2G2A transgenic mice. However, when mice were treated
with inter-peritoneal injection of lipopolysaccharide to
promote inflammation, expression of sPLA2-IIA but not
sPLA2-V was stimulated. Difference in the localization of
these two enzymes in the atherosclerotic plaques of mice and
humans further support a distinct functional divergence (20,25).
Inverse relationship between LDL levels and sPLA2
Surprisingly, compared to PLA2G5 H27 carriers, H1 carriers
had significantly lower LDL cholesterol (P , 0.00004), but
significantly higher oxLDL/LDL (P ¼ 0.006) and borderline
higher sPLA2-IIA mass (P ¼ 0.04), so in part the lower
LDL levels could reflect higher LDL hydrolysis by
sPLA2-IIA. This seems unlikely, since as discussed above,
PLA2G2A haplotypes that had a strong association with
sPLA2-IIA levels showed no effect on LDL cholesterol
levels. PLA2G2A and PLA2G5 lie head to tail on chromosome
1p34 36.1 (2) (in a cluster with other sPLA2 genes), thus it is
Figure 1. (A) Map of the PLA2G5 gene showing the selected tagging SNPs numbered from the start of exon 1. (B) Haploview LD (D
) of tSNPs of both PLA2G5
and PLA2G2A. The darker boxes represent the stronger LD. The D
for any two SNPs is presented in the box representing their intersection. No number indicates
of 1. The head to tail orientation of the two genes and the tSNPs used in this and our previous study (22) are presented.
1440 Human Molecular Gen etics, 2007, Vol. 16, No. 12
by guest on May 29, 2013 from
possible that variation within this intergenic region may be
influencing the expression of both genes. PLA2G5 SNPs
2423G.A and 1742 Gin/del are both associated with signifi-
cant differences in sPLA2-IIA levels (see Supplementary
Material, Tables), and from Figure 1B it is clear that both
these SNPs show significant LD with PLA2G2A 763C. G,
which shows strong statistical significant association with
sPLA2-IIA levels (P , 0.0001) (see Supplementary Material,
Tables in 22). Therefore, it is probable that the association of
PLA2G5 haplotypes with sPLA2-IIA mass (explaining only
0.6% of the sPLA2-IIA variance) is simply reflecting the LD
that exists in this gene cluster. The effect on lipid levels there-
fore suggest that the association of PLA2G5 haplotypes with
LDL levels are due to the haplotypic effects associated with
differences in sPLA2-V activity/mass itself.
Thus, we speculate that, in comparison to haplotypes H2 7,
H1 should be associated with higher sPLA2-V, and since high
sPLA2 activity is reported to increase risk in humans (13) and
mice (5), this would lead to increased CHD risk. On the face
of it this appears counterintuitive, since the lower LDL chol es-
terol levels in H1 carriers, compared to H2 7, should be associ-
ated with decreased risk. However, the lower plasma LDL levels
seen in the H1 carriers could be the result of LDL conversion to
sdLDL particles due to increased phospholipid hydrolysis. This
would result in increased ret ention of LDL in the arterial wall,
increased oxidation and thus lead to increased atherosclerosis.
Since sdLDL is more prone to oxida tion, the oxLDL/LDL
ratio should also be higher. We did not see an effect of
PLA2G5 haplotyp e on sdLDL levels but we did see a
significant effect of haplotypes on oxLDL/LDL. Carriers of
haplotype H1 had oxLDL/LDL levels which were 22% higher
(10.19 U/mmol) compared to H27 (8.35 U/mmol, P ¼ 0.006).
There is support for these associations and the predicted
outcomes from the study by Mallat et al. (13), who measured
both sPLA2 activity and mass in patients with acute coronary
syndrome. While the sPLA2-IIA mass assay shows no cross
Table 3. Inferred haplotype frequencies in UDACS
Haplotype name Haplotype combination Frequency in UDACS
H1 AGGCGinTAdelT 0.359
H2 CGACGdelTAinT 0.169
H3 CGGCGinTAinG 0.128
H4 AGGCGdelTAinT 0.094
H5 AGGCGinTAinT 0.060
H6 CAGTGinTAinT 0.059
H7 CGGTGinTAinT 0.055
AGGTGinTAinT 0.032
CGGCGinTAinT 0.026
CGGCGdelTAinG 0.003
AGGCGinTAinG 0.003
CAGTGinTAinT 0.002
CGACGdelTAdelT 0.002
AGGTGdelTAinT 0.002
CGGTGinTAinT 0.002
AGGTGinTAdelT 0.001
CGGCGinTAdelG 0.001
CAGCGinTAinT 0.001
tSNP order from left to right is: 21437C. A, 21251G. A, 2423G. A,
1640C. T, 1742G in/del, 11609TA in/del and 22507T. G.
Table 4. Association of mean values (95% CI) of intermediate phenotypes with common haplotypes H17 in UDACS
Quantitative traits and
P-values compared to
P-value H1 versus
H2 7 unadjusted
P-value H1 versus
H2 7 adjusted
sPLA2 IIA levels (ng/ml)
2.36 (2.072.69) 1.58 (1.28 1.96) 1.88 (1.47 2.42) 1.89 (1.412.53) 2.23 (1.49 3.36) 2.06 (1.43 2.97) 1.97 (1.20 3.25) 1.46 (0.810.90) 1.85 (0.911.01)
P-value 0.001 0.13 0.19 0.81 0.51 0.50 0.04 0.01
Cholesterol (mmol/l) 2.59 (2.502.67) 2.85 (2.71 3.00) 2.93 (2.76 3.12) 2.97 (2.743.20) 2.67 (2.41 2.94) 2.87 (2.62 3.14) 2.79 (2.52 3.07) 2.64 (2.572.72) 2.87 (2.79 2.94)
P-value 0.008 0.001 0.001 0.59 0.04 0.18 0.00003 0.001
HDL (mmol/l)
0.72 (0.690.75) 0.69 (0.64 0.74) 0.75 (0.70 0.82) 0.76 (0.680.85) 0.64 (0.56 0.73) 0.69 (0.60 0.79) 0.78 (0.66 0.92) 0.64 (0.550.58) 0.72 (0.590.61)
P-value 0.37 0.36 0.42 0.11 0.53 0.35 0.83 0.90
LDL (mmol/l)
1.34 (1.271.42) 1.65 (1.50 1.81) 1.63 (1.46 1.82) 1.65 (1.411.92) 1.48 (1.22 1.79) 1.62 (1.33 1.98) 1.48 (1.22 1.81) 1.61 (0.870.92) 1.61 (0.870.92)
P-value ,0.001 0.01 0.02 0.38 0.07 0.37 0.00004 0.00005
oxLDL/LDL (U/mmol)
9.40 (8.6010.10) 7.8 (6.409.40) 7.60 (5.829.75) 7.20 (5.13 9.71) 9.68 (7.31 12.34) 6.36 (4.588.45) 7.45 (5.55 9.76) 8.35 (7.60 9.14) 7.61 (6.918.35)
P-value 0.1 0.13 0.11 0.87 0.01 0.13 0.006 0.004
Mean value for one copy of each haplotype adjusted for age, gender, CHD status and statin use.
Square root transformation.
Human Molecular Genetics, 2007, Vol. 16, No. 12 1441
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reactivity with sPLA2-V, the activity assay measures most
plasma sPLA2s (i.e. including both -IIA and -V) (13). Those
individuals without hyperlipidaemia had significantly higher
sPLA2 activity compared to those with hyperlipidaemia
(2.88 versus 2.33 nmol/min/ml , respectively; P ¼ 0.001), and
the cumulative incidence of death or MI according to tertiles
of sPLA2 in patients with cholesterol 197 mg/dl was 8.4%
compared to 5.2% in those with cholesterol.197 mg/dl (13).
Thus, this stud y also shows an inverse relationship between
plasma lipids and sPLA2 act ivity (13).
Limitations of the study
The tSNPs identified for this study were derived from the rese-
quenced data from the NIEHS database and have an r
¼ 0.92.
A comparison cannot be made directly to those tSNPs
obtained from the recent HAPMAP release (21). This high-
lights the issue of tSNP identification in a rapidly changing
field where both the databases are constantly upgraded, as
well as the algorithms for tSNP identification. The use of
additional tSNPs might possibly sub-divide haplotype H1,
allowing a clearer identification of the relevant haplotype for
functional studies. However, the use of a limited set of
tSNPs cannot be a confounder of an association that is ident-
ified. In the absence of a specific sPLA2-V assay, we cannot
exclude the possibility that these haplotypic effects on LDL
cholesterol levels are independent of sPLA2-V activity or
mass, and we cannot determine what the association of these
PLA2G5 haplotypes actually are with sPLA2-V levels. In
addition, because of the relatively small sample size, the
UDAC study was insufficiently powered to detect any associ-
ations between PLA2G5 inferred haplotypes and CHD risk.
The association between haplotype H1 and lower LDL
levels has been reported here in patients with T2D, and the
association may not be the same in non-T2D individuals.
Finally, the strong LD across the gene cluster means that it
is not possible to distinguish PLA2G5 and PLA2G2A effects
entirely, although LD may be lower in non-Caucasians,
which could allow for better discrimination of these effects.
The association of PLA2G5 haplotypes with markers of CHD
provides the foundation for further investigations in case-
control and prospective studies, specifically investigating
whether these haplotypes are associated with CHD risk and
with LDL levels in non-diabetic subjects. Despite these limit-
ations, this study represents the first investigation of genetic
variation in PLA2G5 and its association with markers of
CHD and strongly supports the recent studies by Rosengren
et al. (20,25) showing a distinct functional difference
between the two closely related sPLA2 enzymes sPLA2-V
and sPLA2-IIA.
Study design
The University College London Diabetes And Cardiovascular
Disease Study (UDACS) has been described in detail else-
where (26,27). In brief, the UDACS consists of 1014 consecu-
tive subjects recruited from the diabetes clinic at Universi ty
College London Hospitals NHS Trust (UCLH) 2001 02
(629 men; 600 Caucasians with T2D). All patients had dia-
betes according to WHO criteria (28). Analysis was restricted
to the Caucasian subjects with T2D to remove possible hetero-
geneity within the sample.
Clinical measurements
CHD event was recorded if any patient had positive coronary
angiography/angioplasty, coronary artery bypass, cardiac thal-
lium scan, exercise tolerance test, myocardial infarction or symp-
tomatic/treated angina (27). Routine plasma traits were measured
including plasma oxidised LDL (oxLDL) by ELISA (Mercodia,
Uppsala, Sweden), expressed as the ratio of oxLDL divide by
total LDL to generate a specific measure of LDL oxidation
(27,29). Plasma total antioxidant status (TAOS), which is inver-
sely related to oxidative stress, was measured by a photometric
assay (30). The percentage sdLDL is derived from the percentage
of LDL sub-classes I and II from the four sub-classes I IV
obtained by ultra-centrifugation (31). Serum sPLA2-IIA levels
were measured by a commercially available ELISA (Cayman
Chemical Company, Ann Arbor, MI, USA). The intra- and inter-
assay coefficients of variation were 6.0 and 10.3%, respectively.
Full ethical approval was granted by the UCLH NHS trust and all
patients included in the study had given written consent.
DNA extraction, tagging-SNP identification
and genotyping
DNA was extracted using the salting out method (32). Tagging
SNPs were identified using the STRAM algorithm (33) on
the PHASE (34) output from the National Institute of Environ-
mental Health Sciences SNP database website (
Figure 2. The mean LDL-cholesterol levels (95% CI) in subjects with 0 (H1/
H1), 1 (H1/H24) and 2 (H24/H2 4) copies of the H1 and combined H2 4
1442 Human Molecular Gen etics, 2007, Vol. 16, No. 12
by guest on May 29, 2013 from Seven tSNPs of PLA2G5 were
identified (rs11573185, rs2148911, rs11573191, rs11573203,
rs640022, rs11573248, rs622450). All SNPs except rs640022
were genotyped using TaqMan technology (Applied Bio-
sciences, ABI, Warrington, UK). Oligonucleotides and MGB
probes are detailed in Supplementary Material, Table S1. The
rs640022 tSNP was determined using a polymerase chain reac-
tion (PCR) with sense (5
and anti-sense (5
) oligo-
nucleotides flanking the variant of interest. Restriction of the
PCR product with the PvuII enzyme results in fragment sizes
of 166/20 bp in common homozygotes, 186/166/20 bp in hetero-
zygotes and 186 bp in rare homozygotes. Fragments were
resolved using Microtitre Array Diagonal Gel Electrophoresis
(MADGE) (35). Two negative controls were included in each
PCR run.
Statistical methods
HardyWei nberg equilibrium was assessed using THESIAS
(23,24). Linkage disequilibrium (LD) as measured by D
was assessed using Haploview (
mpg/haploview/). All analyses were performed on normally
distributed data after appropriate transformation (log or
square root). Results are presented as mean and sta ndard
deviation (SD). Parametric or non-parametric (Krusk all
Wallis) analysis of variance was used, when appropriate, to
compare the changes of the continuous variables across the
SNPs categories. Adjusted P-values were obtained from the
analysis of covariance for continuous data, and logistic
regression for categorical data. Haplotypes were inferred
using both THESIAS (23,24) and PHASE (34) excluding indi-
viduals with missing values. Th e haplotypic pair for each
subject was calculated by PHASE and only the haplotypes
with frequencies 5% were used for further analysis.
Because of multiple testing, the significance level was taken
as P , 0.01, instead of an inappropriately conservative
Bonferroni-like adjustment of the P-values (36,37).
Supplementary Material is available at HMG Online.
We thank Dr Birit Johansson for helpful discussion and critical
appraisal of the manuscript. We would like to thank contribu-
tors to the University College London Diabetes and Cardio-
vascular Study. P.J.T., P.T.E.W., S.R.T. and S.E.H. are
supported by the British Heart Foundation Grants RG2000/
15 and FS/2002/087/14762 and FS/2004/039. Diabetes UK
supported J.W.S. (BDA: RD01/0001357) and the creation of
UDACS. O.W. is supported by the Swedish Heart and Lung
Foundation Grants 200441654 and 20041243.
Conflict of Interest statement. None declared.
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... Impairment of this process in Pla2g5 −/− mice leads to exacerbation of diet-induced obesity and insulin intolerance, accompanied by elevated phospholipid levels in plasma LDL. This phenotype is reminiscent of clinical evidence that a PLA2G5 polymorphism is associated with plasma LDL levels in patients with type 2 diabetes [56] and that the levels of PLA2G5 mRNA expression in WAT are inversely correlated with plasma LDL levels in obese subjects [41]. sPLA 2 -V is a "Th2-prone sPLA 2 " induced in M2 macrophages by the Th2 cytokines IL-4 and IL-13 and promotes Th2-driven pathologies such as asthma. ...
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The phospholipase A2 (PLA2) superfamily contains more than 50 enzymes in mammals that are subdivided into several distinct families on a structural and biochemical basis. In principle, PLA2 has the capacity to hydrolyze the sn-2 position of glycerophospholipids to release fatty acids and lysophospholipids, yet several enzymes in this superfamily catalyze other reactions rather than or in addition to the PLA2 reaction. PLA2 enzymes play crucial roles in not only the production of lipid mediators, but also membrane remodeling, bioenergetics, and body surface barrier, thereby participating in a number of biological events. Accordingly, disturbance of PLA2-regulated lipid metabolism is often associated with various diseases. This review updates the current state of understanding of the classification, enzymatic properties, and biological functions of various enzymes belonging to the PLA2 superfamily, focusing particularly on the novel roles of PLA2s in vivo.
... Dans les désordres du métabolisme, comme l'obésité, cette enzyme joue un rôle protecteur en hydrolysant la PC des LDL et en établissant un équilibre immunitaire qui empêche l'inflammation du tissu adipeux (Sato et al., 2014). Chez l'Homme, les polymorphismes du gène Pla2g5 sont associés à des niveaux différents de LDL chez les patients présentant un diabète de type 2 (Wootton et al., 2007). ...
Le paludisme est une maladie tropicale causée par un parasite du genre Plasmodium. Chez l’Homme, un niveau élevé de phospholipase A2 sécrétée de groupe IIA humaine (hGIIA) est mesuré dans le plasma des patients impaludés. Cette enzyme est connue pour son rôle antibactérien et pro-inflammatoire. Cependant, son rôle dans le paludisme n’a jamais été exploré. Pour comprendre le rôle in vivo de la hGIIA dans cette pathologie, nous avons entrepris la caractérisation hématologique, histopathologique et immunohistochimique de l’infection de souris C57BL/6, transgéniques (Tg+) pour l’enzyme humaine, par l’espèce murine Plasmodium chabaudi chabaudi 864VD. Ce modèle reproduit un paludisme non létal. Nos résultats ont permis d’établir que les souris Tg+ ont un meilleur contrôle de l’infection au moment du pic de crise parasitaire (J14 post-inoculation), avec une diminution de 27% de la parasitémie, comparé aux souris « littermates » non transgéniques (Tg-). L’injection de hGIIA recombinante aux jours 12, 13 et 14 p.i. (0,125 mg/kg deux fois par jour) à des souris C57BL/6 wild-type (WT) infectées par P. c. chabaudi 864VD provoque une diminution d’environ 19% de la parasitémie à J14 p.i., démontrant un rôle direct de la hGIIA dans la diminution de la population parasitaire. Les données hématologiques montrent que l’infection chez la souris Tg+ provoque une anémie plus durable que chez la souris Tg- et une élévation nettement plus importante du nombre de leucocytes, en particulier des polynucléaires neutrophiles. Chez la souris Tg+ parasitée, on observe aussi l’activation d’un nombre important de lymphocytes et une activation spécifique des monocytes avant le pic de crise. Chez la souris Tg- infectée, les données histologiques mettent en avant une meilleure récupération des lésions histopathologiques du foie et une hyperplasie des lymphocytes B dans la rate, tandis que les souris Tg+ infectées présentent des lésions hépatiques tardives et une hématopoïèse extramédullaire splénique. Les résultats des analyses par RT-qPCR suggèrent que l’ARNm de la hGIIA augmente au pic parasitaire dans le foie des souris Tg+ infectées, mais diminue dans la rate et les cellules sanguines. L’injection de hGIIA recombinante au début de la phase patente est sans effet sur la parasitémie, ce qui laisse supposer que des événements plus tardifs dans l’infection sont nécessaires à l’activité antiparasitaire de l’enzyme. L’étude du rôle des lipoprotéines oxydées comme substrat potentiel de l’activité antiparasitaire de l’enzyme, basée sur des résultats in vitro, est abordée. En conclusion, nos études ont permis de dresser un tableau large de l’infection à Plasmodium chez la souris exprimant la hGIIA, et ouvrent de nouvelles perspectives dans l’analyse du rôle de l’enzyme dans la physiopathologie du paludisme.
... This was affirmed by treating the group V transgenic mice with broad spectrum sPLA2 inhibitors. There is an evidence for accumulation of its product involved in the type II diabetes (Wootton et al. 2007) and many metabolic disorders. The group X sPLA2s are not in concurrent with V. ...
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The resurgence of resistance to inhibitors in insects has necessitated the search for newer and more effective biological means of controlling insects. Inhibitors from diverse sources are being explored for understanding their inhibitory potential. Among the alternatives, protease inhibitors represent an attractive class of biopesticides with several important features. Such protease inhibitors are environmentally friendly to work as defense molecules against plant pest and pathogen. Helicoverpa armigera is a voracious feeder blessed with the destructive potential of crop plants. These “eating machines” have robust metabolic processes and exhibit much faster growth and development. Targeting digestive enzymes and/or chitin present in the peritrophic membrane, a film like structure that separates food from the gut tissue of many insect pests is gaining emphasis in alternative pest control strategies. This article presents some recently explored proteins that exhibit enormous potential in biocontrol of insect pests.
... To determine whether decreased intestinal lipid absorption might be caused by changes in expression of lipid processing and transporting molecules, we performed transcriptome analyses of total RNA samples isolated from the small intestines of HFD-fed WT type and dKO animals. Cluster analyses of lipid transport-related gene transcripts revealed differences between WT type and dKO animals in the expression of adipogenesis regulators, such as peroxisome proliferator-activated receptors γ (PPARγ) and  48 , fatty acid binding proteins 1 and 2 (FABP1, 2) 49 , cytoplasmic fatty acid chaperones expressed in adipocytes, acyl-coenzyme A synthetases 1 and 4 (ACSL1,4) found to be associated with histone acetylation in adipocytes, lipid loading and insulin sensitivity 50 , SLC27a1, a2 fatty acid transport proteins, critical mediators of fatty acid metabolism 51 , angiotensin-converting enzyme (ACE) playing a regulatory role in adipogenesis and insulin resistance 52 , CROT, a carnitine acyltransferase important for the oxidation of fatty acids, a critical step in their metabolism 53 , phospholipase PLA2G5 robustly induced in adipocytes of obese mice [54][55][56] . Upon those, we decided to study in more detail the regulation of CD36 that encodes a very long chain fatty acids (VLCFA) transporter. ...
Adipogenesis is a complex process governed by multiple signaling cascades operating through various regulatory factors and specific downstream genes. Here we identified TIS7 (IFRD1) and its orthologue SKMc15 (IFRD2) as new regulators of adipogenesis and fat metabolism. Mice deficient in both TIS7 and SKMc15 (dKO) had severely reduced adipose tissue and were resistant to diet-induced obesity. Wnt signaling, a negative regulator of adipocyte differentiation, was significantly up regulated in TIS7 SKMc15 dKO mice. Elevated levels of the Wnt/β-catenin target protein Dlk-1 inhibited the expression of adipogenesis regulators PPARγ and C/EBPα, and fatty acid transporter CD36. Although both, TIS7 and SKMc15, contributed to this phenotype, TIS7 acted by controlling Wnt signaling and SKMc15 utilized a distinct mechanism. Our study suggests that TIS7 and SKMc15 play an important role in the control of lipid uptake and contribute to the regulation of adipocyte differentiation.
... These findings are in agreement with a recent study where genotyping of 1386 CHD cases and 3532 controls was done, as well as meta-analysis using data from several studies such as the WTCCC [6], CVHS GWAS, Northwick Park Heart Study II (NPHS II), Simon Broome Study, HIFMECH, CABG and UDACS was performed [2,7,8]. The meta-analysis obtained an OR of 1.16, 95% CI 1.05-1.29 indicating statistically significant association of the SNP rs7025486 (A) with CAD. ...
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Genome wide association study has identified rs7025486 G>A polymorphism within DAB2IP (Disabled homolog 2-interacting protein) gene with increased risk of coronary heart disease (CAD). In this study we have determined the frequency and association of rs7025486 with CAD in Indians. The study was performed on 214 patients with CAD and 125 controls. The ‘AA’ genotype was associated with an increased risk in the CAD age group <50 yrs as compared to CAD age group>50 yrs (OR 3.149; P 0.034) and controls >50 yrs (OR 3.430; P 0.080). The risk allele (A) was significantly associated with premature CAD.
... 212, 1901Med. 212,-1919 7) Miki, Y., Yamamoto, K., Taketomi, Y., Sato, H., Shimo, K., Kobayashi, T., Ishikawa, Y., Ishii, T., 190 Benign fleck retina 191 Nakanishi, H., Ikeda, K., Taguchi, R., Kabashima, K., Arita, M., Arai, H., Lambeau, G., Bollinger, J.M., Hara, S., Gelb, M.H. and Murakami, M. (2013) Lymphoid tissue phospholipase A 2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators. J. Exp. ...
The phospholipase A2 (PLA2) family comprises a group of lipolytic enzymes that typically hydrolyze the sn-2 position of glycerophospholipids to give rise to fatty acids and lysophospholipids. The mammalian genome encodes more than 50 PLA2s or related enzymes, which are classified into several subfamilies on the basis of their structures and functions. From a general viewpoint, the PLA2 family has mainly been implicated in signal transduction, producing bioactive lipid mediators derived from fatty acids and lysophospholipids. Recent evidence indicates that PLA2s also contribute to phospholipid remodeling for membrane homeostasis or energy production for fatty acid β-oxidation. Accordingly, PLA2 enzymes can be regarded as one of the key regulators of the quality of lipids, which I herein refer to as lipoquality. Disturbance of PLA2-regulated lipoquality hampers tissue and cellular homeostasis and can be linked to various diseases. Here I overview the current state of understanding of the classification, enzymatic properties, and physiological functions of the PLA2 family.
... The metabolic regulation by PLA2G5 is illustrated in Fig. 7D. Of note, PLA2G5 expression in human adipose tissue inversely correlates with plasma LDL levels and PLA2G5 mutations are associated with LDL levels in patients with type 2 diabetes or obesity (Sergouniotis et al., 2011;Wootton et al., 2007), implying the relevance of these findings to humans. ...
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Within the phospholipase A 2 (PLA 2) superfamily that hydrolyzes phospholipids to yield fatty acids and lysophospholipids, the secreted PLA 2 (sPLA 2) enzymes comprise the largest family that contains 11 isoforms in mammals. Individual sPLA 2 s exhibit unique distributions and specific enzymatic properties, suggesting their distinct biological roles. While sPLA 2 s have long been implicated in inflammation and atherosclerosis, it has become evident that they are involved in diverse biological events through lipid mediator-dependent or mediator-independent processes in a given microenvironment. In recent years, new biological aspects of sPLA 2 s have been revealed using their transgenic and knockout mouse models in combination with mass spectrometric lipidomics to unveil their target substrates and products in vivo. In this review, we summarize our current knowledge of the roles of sPLA 2 s in metabolic disorders including obesity, hepatic steatosis, diabetes, insulin resistance, and adipose tissue inflammation.
... The metabolic regulation by PLA2G5 is illustrated in Fig. 7D. Of note, PLA2G5 expression in human adipose tissue inversely correlates with plasma LDL levels and PLA2G5 mutations are associated with LDL levels in patients with type 2 diabetes or obesity (Sergouniotis et al., 2011;Wootton et al., 2007), implying the relevance of these findings to humans. ...
Within the phospholipase A2 (PLA2) family that hydrolyzes phospholipids to yield fatty acids and lysophospholipids, secreted PLA2 (sPLA2) enzymes comprise the largest group containing 11 isoforms in mammals. Individual sPLA2s exhibit unique tissue or cellular distributions and enzymatic properties, suggesting their distinct biological roles. Although PLA2 enzymes, particularly cytosolic PLA2 (cPLA2α), have long been implicated in inflammation by driving arachidonic acid metabolism, the precise biological roles of sPLA2s have remained a mystery over the last few decades. Recent studies employing mice gene-manipulated for individual sPLA2s, in combination with mass spectrometric lipidomics to identify their target substrates and products in vivo, have revealed their roles in diverse biological events, including immunity and associated disorders, through lipid mediator-dependent or -independent processes in given microenvironments. In this review, we summarize our current knowledge of the roles of sPLA2s in various immune responses and associated diseases.
Inulin, as a dietary fiber, exerted prominent anti-obesity effects by modulating gut microbiota. However, the possible relationship and interplay of gut microbiome and function of distal intestine is still unclear now. This study aimed to investigate the possible targets of microbes and the related intestinal genes mediated by inulin. C57 BL/6 male mice were randomly allocated to chow diet (Chow) group, high-fat diet (HFD) group, and HFD supplemented with 3 % inulin (Inulin) group. Compared with HFD treatment, inulin supplementation significantly decreased the body weight, fat deposition, and fasting blood glucose level. In addition, mice treated with inulin had a remarkable alteration in the structure of cecal microbiota and transcriptomic profiling of ileum. In particular, inulin supplementation significantly reversed the HFD induced expression of Bacteroides, Allobaculum and nonrank_f_Bacteroidates_S24-7_group, and reversed the expression of genes belonging to phospholipase A2 (PLA2) family and cytochrome P450 (CYP450) family. In summary, inulin might alleviate HFD-induced fat deposition and metabolic disorders via regulating lipid metabolism of ileum, while the interaction between the sPLA2s and gut microbes might play important roles in the process.
Secreted phospholipase A2 (sPLA2) is an enzyme that hydrolyzes the phospholipids at a specific site and initiates the inflammatory/arachidonic acid (AA) pathway. Snake venom sPLA2s are neuro- and myo- toxins while different tissues of human body produce sPLA2s involved in inflammation, signalling and atherosclerosis. These wide ranges of pharmacological activities exerted by sPLA2s with a conserved structural domain thrust scientists to understand the molecular mechanism behind it, in the past two decades. This resulted in availability of well characterized biological data on function of sPLA2s of almost all types and continuous deposition of their structural data in the protein data bank. Additionally, the isoform specific inhibitors can also be retrieved from different chemical compound databases for futuristic and knowledge-based computational studies. Hence, inhibition or regulation of sPLA2s deserves a significant clinical and pharmacological importance, despite their anti-bacterial and anti-viral activities. In the present situation with increase in snake bite or envenomation cases worldwide, particularly in tropical countries, further studies on catalytic and inhibition mechanisms of sPLA2 isoforms at atomic details are necessary to confront the associated pathologies. This chapter is structured to provide detailed information of the current knowledge on the sPLA2 biology and future perspectives.
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The presence of a lipoprotein profile with abundance of small, dense low density lipoproteins (LDL), low levels of high density lipoproteins (HDL), and elevated levels of triglyceride-rich very low density lipoproteins is associated with an increased risk for coronary heart disease. The atherogenicity of small, dense LDL is believed to be one of the main reasons for this association. This particle contains less phospholipids (PL) and unesterified cholesterol than large LDL, and the apoB-100 appears to occupy a more extensive area at its surface. Although there are experiments that suggest a metabolic pathway leading to the overproduction of small, dense LDL, no clear molecular model exists to explain its association with atherogenesis. A current hypothesis is that small, dense LDL, because of its higher affinity for proteoglycans, is entrapped in the intima extracellular matrix and is more susceptible to oxidative modifications than large LDL. Here we describe how a specific reduction of approximately 50% of the PL of a normal buoyant LDL by immobilized phospholipase A2(PLA2) (EC produces smaller and denser particles without inducing significant lipoprotein aggregation (<5%). These smaller LDL particles display a higher tendency to form nonsoluble complexes with proteoglycans and glycosaminoglycans than the parent LDL. Binding parameters of LDL and glycosaminoglycans and proteoglycans produced by human arterial smooth muscle cells were measured at near to physiological conditions. The PLA2-modified LDL has about 2 times higher affinity for the sulfated polysaccharides than control LDL. In addition, incubation of human plasma in the presence of PLA2 generated smaller LDL and HDL particles compared with the control plasma incubated without PLA2. These in vitro results indicate that the reduction of surface PL characteristic of small, dense LDL subfractions, besides contributing to its small size and density, may enhance its tendency to be retained by proteoglycans.
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Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 μM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 μM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.
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Density gradient ultracentrifugation of low density lipoproteins (LDL) from 12 normal subjects showed multiple, distinct isopycnic bands. Densitometric scanning of the gradient tubes revealed that each band could be assigned to one of four density intervals and that the boundaries of these intervals were consistent among all the subjects. Analytic ultracentrifuge flotation (S(f)(0)) rates were assigned to the four density intervals, and there was a strong correlation between peak S(f)(0) rate and peak isopycnic banding position (R(f)) of the LDL in the 12 subjects. The S(f)(0) value corresponding to the boundary between the two most buoyant LDL density subgroups was 7.5. This value is close to that previously demonstrated to define two LDL subdivisions (S(f)(0) 0-7 and S(f)(0) 7-12) that were discriminated by differing concentrations in men and women, and differing statistical relationships with levels of HDL and VLDL in a normal population. Further delineation of distinct subspecies of LDL was afforded by electrophoresis in 2-16% gradient polyacrylamide gels. Densitometric scans of protein-stained gels revealed multiple peaks, and particle diameters were assigned to these peaks using calibration markers. Particles of diameter >/= 280 A included both IDL and Lp(a), the latter defined by pre-beta mobility on agarose electrophoresis and density > 1.050 g/ml. LDL particles with diameters 220-272 A could be grouped into seven size intervals defined by modes in the distribution of gradient gel electrophoretic peaks in LDL from a group of 68 healthy men and women. Particle diameters of the major peaks in each of seven density subfractions decreased with increasing density of the fractions. However, particles within each of the size groups were distributed across a range of densities. Use of a lipid-staining procedure allowed identification of electrophoretic bands in whole plasma which corresponded to those seen in isolated LDL, eliminating the possibility that ultracentrifugation was responsible for formation of the subspecies detected by the gradient gel procedure. The application of density gradient ultracentrifugation and gradient gel electrophoresis provides a means of characterizing LDL from normal humans in terms of multiple distinct subpopulations which may also prove to have differing metabolic and pathologic properties.-Krauss, R. M., and D. J. Burke. Identification of multiple subclasses of plasma low density lipoproteins in normal humans.
We investigate the contribution of the Iberian bat fauna to the cryptic diversity in Europe using mitochondrial (cytb and ND1) and nuclear (RAG2) DNA sequences. For each of the 28 bat species known for Iberia, samples covering a wide geographic range within Spain were compared to samples from the rest of Europe. In this general screening, almost 20% of the Iberian species showed important mitochondrial discontinuities (K2P distance values > 5%) either within the Iberian or between Iberian and other European samples. Within Eptesicus serotinus and Myotis nattereri, levels of genetic divergence between lineages exceeded 16%, indicating that these taxa represent a complex of several biological species. Other well-differentiated lineages (K2P distances between 5–10%) appeared within Hypsugo savii, Pipistrellus kuhlii and Plecotus auritus, suggesting the existence of further cryptic diversity. Most unsuspected lineages seem restricted to Iberia, although two have crossed the Pyrenees to reach, at leas...
A series of copolymers of predominantly poly(ethylene oxide) (PEO) with mono-phenyl (HQ), biphenyl (BP) units, or both of them (HQ/BP) in the backbone were synthesized. The solid polymer electrolytes (SPEs) were prepared from three different types of copolymers (HQ-PEG, BP-PEG, and HQ/BP-PEG) employing lithium perchlorate (LiClO4) as a lithium salt at a fixed salt concentration of [EO]/[Li+]=8. Their ionic conductivities were investigated to exploit the structure–ionic conductivity relationships as a function of structural change in rigid phenyl units and chain length ratio between flexible PEO chain and rigid phenyl units. As more rigid phenyl units were incorporated in the backbone chain, the formation inter- and intra-molecular complex with LiClO4 became weaker and lower ionic conductivities were observed. And it was also found that higher ionic conductivity is obtained with increasing PEO chain length because inter- and intra-molecular dissociation power of PEO increases.
Secretory phospholipase A(2) (PLA(2)) Can be proatherogenic both in the circulation and in the arterial wall. In blood plasma, PLA(2) can modify the circulating lipoproteins and so induce formation of small dense LDL particles, which are associated with increased risk for cardiovascular disease. In the arterial wall, PLA(2) can hydrolyze lipoproteins. The PLA(2)-modified lipoproteins bind tightly to extracellular proteoglycans, which may lead to their enhanced retention in the arterial wall. The modified lipoproteins may also aggregate and fuse, which can lead to accumulation of their lipids within the extracellular matrix. The PLA(2)-modified particles are more susceptible to further modifications by other enzymes and agents and can be taken up by macrophages, leading to accumulation of intracellular lipids. In addition, lysophospholipids and free fatty acids, the hydrolysis products of PLA(2), promote atherogenesis. Thus, these lipid mediators can be carried, either by the PLA(2)-modified lipoproteins themselves or by albumin, into the arterial cells, which then undergo functional alterations. This may, in turn, lead to specific changes in the extracellular matrix, which increase the retention and accumulation of lipoproteins within the matrix. In the present article, we discuss the possible actions of PLA(2) enzymes, especially PLA(2)-IIA, in the arterial wall during atherogenesis.
The Group IIA phospholipase gene (PLA2G2A) protein coding regions exhibit significant homology with recently described Group IIC (PLA2G2C) and Group V (PLA2GV) genes. All three genes are present in many mammalian species and are expressed in a tissue-specific pattern. Here, we demonstrate in human that they are tightly linked and map to chromosome 1p34–p36.1. We also show that the homologous mouse loci are tightly linked (no observed recombination) on the distal part of chromosome 4, a region exhibiting synteny with human 1p34–p36. Unlike its rodent counterpart, humanPLA2G2Cappears to be a nonfunctional pseudogene.
Adjustments for making multiple comparisons in large bodies of data are recommended to avoid rejecting the null hypothesis too readily. Unfortunately, reducing the type I error for null associations increases the type II error for those associations that are not null. The theoretical basis for advocating a routine adjustment for multiple comparisons is the "universal null hypothesis" that "chance" serves as the first-order explanation for observed phenomena. This hypothesis undermines the basic premises of empirical research, which holds that nature follows regular laws that may be studied through observations. A policy of not making adjustments for multiple comparisons is preferable because it will lead to fewer errors of interpretation when the data under evaluation are not random numbers but actual observations on nature. Furthermore, scientists should not be so reluctant to explore leads that may turn out to be wrong that they penalize themselves by missing possibly important findings.
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