CXCL5 polymorphisms are associated with variable blood pressure in cardiovascular disease-free adults

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Title:
CXCL5 polymorphisms are associated with variable blood pressure in cardiovascular disease-free adults
Physical Description:
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Language:
English
Creator:
Beitelshees, Amber L
Aquilante, Christina L
Allayee, Hooman
Langaee, Taimour Y.
Welder, Gregory J.
Schofield, Richard S.
Zineh, Issam
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BioMed Central
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Abstract:
Objective: Leukocyte count has been associated with blood pressure, hypertension, and hypertensive complications. We hypothesized that polymorphisms in the CXCL5 gene, which encodes the neutrophilic chemokine ENA-78, are associated with blood pressure in cardiovascular disease (CVD)-free adults and that these polymorphisms are functional. Methods and results: A total of 192 community-dwelling participants without CVD or risk equivalents were enrolled. Two CXCL5 polymorphisms (−156 G > C (rs352046) and 398 G > A (rs425535)) were tested for associations with blood pressure. Allele-specific mRNA expression in leukocytes was also measured to determine whether heterozygosity was associated with allelic expression imbalance. In −156 C variant carriers, systolic blood pressure (SBP) was 7 mmHg higher than in −156 G/G wild-type homozygotes (131 ± 17 vs. 124 ± 14 mmHg; P = 0.008). Similarly, diastolic blood pressure (DBP) was 4 mmHg higher in −156 C variant carriers (78 ± 11 vs. 74 ± 11 mmHg; P = 0.013). In multivariate analysis of SBP, age, sex, body mass index, and the −156 G > C polymorphism were identified as significant variables. Age, sex, and the −156 G > C SNP were further associated with DBP, along with white blood cells. Allelic expression imbalance and significantly higher circulating ENA-78 concentrations were noted for variant carriers. Conclusion: CXCL5 gene polymorphisms are functional and associated with variable blood pressure in CVD-free individuals. The role of CXCL5 as a hypertension- and CVD-susceptibility gene should be further explored. Keywords: CXCL5, ENA-78, Blood pressure, Hypertension, Leukocytes
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doi:10.1186/1479-7364-6-9 Cite this article as: Beitelshees et al.: CXCL5 polymorphisms are associated with variable blood pressure in cardiovascular disease-free adults. Human Genomics 2012 6:9.
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Beitelshees et al. Human Genomics 2012, 6:9
http://www.humgenomics.com/content/6/1/9


SHuman Genomics


CXCL5 polymorphisms are associated with

variable blood pressure in cardiovascular

disease-free adults

Amber L Beitelshees*t, Christina L Aquilante2t, Hooman Allayee3, Taimour Y Langaee4, Gregory J Welder4,
Richard S Schofield5 and Issam Zineh4


Abstract
Objective: Leukocyte count has been associated with blood pressure, hypertension, and hypertensive
complications. We hypothesized that polymorphisms in the CXCL5 gene, which encodes the neutrophilic
chemokine ENA-78, are associated with blood pressure in cardiovascular disease (CVD)-free adults and that
these polymorphisms are functional.
Methods and results: A total of 192 community-dwelling participants without CVD or risk equivalents were
enrolled. Two CXCL5 polymorphisms (-156 G > C (rs352046) and 398 G> A (rs425535)) were tested for associations
with blood pressure. Allele-specific mRNA expression in leukocytes was also measured to determine whether
heterozygosity was associated with allelic expression imbalance. In -156 C variant carriers, systolic blood pressure
(SBP) was 7 mmHg higher than in -156 G/G wild-type homozygotes (131 17 vs. 124 14 mmHg; P= 0.008).
Similarly, diastolic blood pressure (DBP) was 4 mmHg higher in -156 C variant carriers (78 + 11 vs. 74 + 11 mmHg;
P= 0.013). In multivariate analysis of SBP, age, sex, body mass index, and the -156 G > C polymorphism were
identified as significant variables. Age, sex, and the -156 G > C SNP were further associated with DBP, along with
white blood cells. Allelic expression imbalance and significantly higher circulating ENA-78 concentrations were
noted for variant carriers.
Conclusion: CXCL5 gene polymorphisms are functional and associated with variable blood pressure in CVD-free
individuals. The role of CXCL5 as a hypertension and CVD-susceptibility gene should be further explored.
Keywords: CXCL5, ENA-78, Blood pressure, Hypertension, Leukocytes


Introduction
The relationship between inflammation and elevated blood
pressure is increasingly being evaluated [1,2]. It has been
shown that elevated concentrations of prototypical pro-
inflammatory markers such as interleukin-6, C-reactive
protein (CRP), and tumor necrosis factor-alpha are
associated with increased blood pressure, incidence of
hypertension, and the likelihood for hypertensive com-
plications [3-14]. It has been further suggested that
this inflammatory-hypertensive relationship results from
increased number or activity of common cellular

'Correspondence abeitels@medicine umaryland edu
Equal contributors
DDivision of Endocrinology, Diabetes and Nutrition, University of Maryland
School of Medicine, 660 W Redwood St, HH469, Baltimore, MD 21201, USA
Full list of author information is available at the end of the article


mediators such as white blood cells (WBC) [15,16]. For
example, studies have demonstrated elevated WBC count
to be associated with increased incident hypertension as
well as increased blood pressure within the normal to pre-
hypertensive range [17-22].
Although the exact mechanistic relationship between
leukocytosis and elevated blood pressure is unknown,
it is plausible that low-grade inflammation may be a
contributing factor. In this regard, WBC count may be a
surrogate marker for increased activation of inflammatory
pathways that cause leukocyte recruitment and activa-
tion. As such, increased activity of leukocytic chemokines
could be related to increased blood pressure.
Epithelial neutrophil activator-78 (ENA-78), a key
leukocytic chemokine that is both a neutrophil attractor


S 2012 Beltelshees et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Biole led Central Creative Commons Attribution License (http//creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.






Beitelshees et al Human Genomics 2012, 6:9
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and activator, has been implicated in many diseases with
an inflammatory component (e.g., obesity, diabetes, sub-
clinical atherosclerosis, acute coronary syndromes) [23-32].
We have previously reported that two single nucleotide
polymorphisms T-!r. -156 G>C (rs352046) and 398
G >A (rs425535), in the gene encoding ENA-78 (CXCL5)
occur in sites important for transcription and exon spli-
cing [33]. In our previous work, a relationship existed
between these SNPs and both plasma concentrations and
leukocyte production of the ENA-78 chemokine protein
[33]. We then went on to show an association between
the CXCL5 -156 G > C polymorphism and worse out-
comes in patients with acute coronary syndromes [27].
In the present work, to the extent that ENA-78 is
important in neutrophil recruitment and degranulation,
we hypothesized that one or both of these polymorph-
isms (-156 G>C and 398 G>A) could be associated
with differences in blood pressure in individuals without
.I. i: .: cardiovascular disease (CVD). .'*.. i ,i ,
we hypothesized that relatively young individuals without
known CVD who were carriers of CXCL5 variant alleles
would exhibit higher systolic blood pressure (SBP), dia-
stolic blood pressure (DBP), or pulse pressure (PP) than
wild-type homozygotes. Furthermore, to assess whether
there was a functional role for these polymorphisms,
we measured .ii. '1 ... I', mRNA expression of CXCL5
in leukocytes obtained from CVD-free individuals who
were heterozygous for the SNPs at both loci.


Materials and methods
Study population
The study population has been previously I. -1.. I
[33]. Briefly, participants were recruited from two sites
in the USA and had to be at least 18 years of age with-
out known CVD or CVD-risk equivalents (e.g., diabetes,
peripheral vascular disease, 10-year Framingham Risk
>20%) as ii. i(i. I by National Cholesterol Education Pro-
gram criteria [34]. Other exclusions were pregnancy,
malignancy, substance abuse, and routine use of medica-
tions known to affect WBC counts such as systemic
steroids and other anti-inflammatory agents. Individuals
were excluded from analysis if they were taking anti-
hypertensive medications for either cardiovascular or
non-cardiovascular indications (e.g., migraine). For blood
pressure measurement, subjects were seated for at least
5 min in a quiet, temperature- ...!. II .1 General Clin-
ical Research Center (GCRC) outpatient clinic room,
and two blood pressure measurements were taken at
least 5 min apart. The average of the dl'.,;.. -(,n blood
pressure measurements was used for this investigation.
Blood samples were obtained from participants enrolled
in University of Florida- and Colorado Multiple Institu-
tional Review Board ,ri .': i l.,'oved studies. All subjects


provided written informed consent to specimen and data
use in genetic association and related studies.

Genotype and inflammatory biomarker determination
Genomic DNA was isolated from whole blood or buccal
cells using previously i :,i.. methods [' ] CXCLS
genotypes were determined by polymerase chain reac-
tion (PCR) and pyrosequencing (Qiagen, Valencia, CA,
USA) as we have previously described [36]. Circulating
high-sensitivity CRP (as a non-specific marker of inflam-
mation) was measured by the Shands Hospital Labo-
ratory at the University of Florida and University
of Colorado GCRC. ENA-78 concentrations were mea-
sured by cytometric fluorescence detection as previously
i. ...i..! (LuminexTM100 IS system; Luminex Corp.,
Austin, TX, USA; Fluorokine' MAP Multiplex Human
Cytokine Panel A; R&D Systems, Minneapolis, MN,
USA) [37]. Samples were stored at -80C until CRP and
ENA-78 detection was performed.

Allele-specific mRNA quantification
To determine whether variant carrier status results in
functional changes at the transcriptional level, we quan-
tified allele-specific mRNA transcripts from leukocytes
using pyrosequencing-based methodology --' "-']. Spe-
i i.. 1i, the presence or absence of allelic expression
imbalance was determined using leukocytes obtained
from 18 individuals who were heterozygotic for both
the -156 G>C and 398 G>A polymorphisms. The
398 G > A SNP was chosen as the genetic biomarker in
these experiments because it is located in the ....: i, re-
gion of CXCL5, while -156 G > C is a promoter poly-
morphism and as such cannot be quantified at the
mRNA level. Because of the near complete linkage of
the studied SNPs, we chose individuals who were het-
erozygotes at both loci so that 398 G > A genotype might
serve as a functional surrogate for the upstream pro-
moter locus.
Leukocyte mRNA was prepared from approximately
6 x 106 cells from each individual using the RNeasy mini
kit (Qiagen, Valencia, CA, USA). Cells were rinsed, lysed,
and homogenized in buffered solutions and subsequently
passed through the RNeasy mini column (Qiagen,
Valencia, CA, USA). Following a series of washes at
room temperature and 15-min incubation with DNase,
concentrations were determined by spectrophotometry
(NanoDrop Technologies, Wilmington, DE, USA).
cDNA was synthesized using approximately 450 ng of
cellular RNA from each individual using a High-
Capacity cDNA Archive Kit (Applied Biosystems, Foster
City, CA, USA) per protocol. Conditions for reverse
transcription were 25C for 10 min :, i,,.. _.-:1 by 37C for
2 h. cDNA quality was assessed by comparing cDNA
and DNA PCR products generated using intron-


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spanning primers by gel electrophoresis. For allele-
i... :'" transcript i in. ,.. ...::. subject DNA and
cDNA underwent PCR simultaneously using previously
S.. Id.. .: (..i..l.1 ,..,. [36]. PCR products obtained for
genotype determination (DNA) and transcript quantifica-
tion (mRNA) were assayed in 1 11 ii, i pyrosequencing
reactions to minimize cycle variability. 'P. '. .. : in. i. 1 i-
analyses were performed in duplicate on three separate
PCR amplification products, and the results were pooled
for analysis. Peak heights were determined by the pyrose-
quencing allele :il.. !ii, ,. : i algorithm. In genomic
DNA, the ratio of 398A:G alleles for DNA in heterozy-
gotes is expected to be approximately 1, whereas signifi-
cant deviations from this ratio in mRNA would suggest
allele expression imbalance associated with the variant
allele.

Statistical analyses
Genotype frequencies were determined by allele count-
ing, and departures from Hardy-:'". ii1.. 1. i.i-,i.,iii:i
were assessed by chi-square analyses. Differences in
blood pressure by genotype groups (0, homozygous for
common allele; 1, heterozygous or homozygous for vari-
ant allele) were compared using one-way ANOVA.
Based on the preexisting sample size and prevalence of
variant alleles, we had 80% power with a two-sided a of
0.05 to detect a 6-mmHg .1.1'. i !!.." in SBP, 4-mmHg
_:-irt ..-..... in DBP, and 4-mmHg ::it.r ..... ..in PP be-
tween genotype _i,,,. Multiple regression analysis was
performed if blood pressure .hfi. .' were seen across
genotype groups. Covariates for multiple regression were
chosen through univariate analyses of age, sex, smoking
status (0, non-smoker; 1, current smoker), body mass
index (BMI), CRP concentration, ENA-78 concentration,
and WBC count. Any variable with a P< 0.1 on univari-
ate analysis was entered into the multivariable model.
Because of small numbers of individuals within racial
groups, analyses could not be performed within racial
strata. However, race (0, white; 1, non-white) was
included in all multivariable analyses, and a race-by-
genotype interaction term was considered in the regres-
sion models to avoid spurious associations secondary to
racial .ill. i. i. in allele frequency. Multiple regression
using step-type selection methods was performed to
determine the joint effects of CXCL5 genotypes and clin-
ical variables on SBP, DBP, or PP. All statistical analyses
were performed using SPSS (version 11.5, SPSS Inc.,
Chicago, IL, USA) or SAS (version 9.1, SAS Institute
Inc., Cary, NC, USA). A P value <0.05 was considered
statistically significant.

Results
Baseline demographic characteristics are shown in
Table 1. Participants were on average 39 + 12 years old


Table 1 Baseline characteristics
Characteristic
Age (mean SD, years)
Women (number
Race/ethnicity (number
White
Black
Hispanic
Other
Family heart disease history (number
Smoking (number
Body mass index (mean SD, kg/m2)
Blood pressure (mean SD, mmHg)


N=192
3912
124 (65)


148(7,
12 (6)
19 (10)
13 (7)
29 (15.
35 (18)
29.6+,


Pulse pressure (mean SD, mmHg) 51 10
Cholesterol (mean SD, mg/dL )
Total 201 +43
LDL 118+36
HDL 55+17
Triglycerides 139 107
White blood cell count, (mean +SD, x09 cells/L) 6.3+2.0
C-reactive protein (median (range), mg/Lb) 1.78 (0.1-16.9)
ENA-78 (median (range), pg/mLb) 362 (32.2-3970)
'Total, HDL, and triglycerides available in 94% of subjects; LDL available in
92% of subjects. bCRP and ENA-78 available for 88% and 91% of subjects,
respectively.

with blood pressures of 126/75 + 15/11 mm Hg. -156
G > C and 398 G >A genotypes were determined for 189
and 188 of the 192 individuals, respectively. The overall
-156 C and 398A minor allele frequencies were both
15%. Variant allele frequencies differed by race whereby
the -156 C allele frequency was : :' .' and 11%,
and 398A allele frequency was 13%, 46%, and 9% in
Caucasians, blacks, and non-black Hispanics, respect-
ively. Genotype distributions satisfied criteria for Hardy-
Weinberg equilibrium (data not shown). The two SNPs
were in a high degree of linkage i.' .,.,-i.i ....... with r2
for Caucasian, black, and Hispanic individuals of 0.82,
1.0, and 0.51, respectively, in our study population.

Genotype association with blood pressure
In -156 C variant carriers, SBP was 7-mmHg 1.:.1.
than in -156 G/G wild-type homozygotes (131+ 17 vs.
124+ 14 mmHg; P= 0.008). Similarly, DBP was 1-..,-.c I
higher in -156 C variant carriers (78+11 vs. 74 11
mmHg; P=0.013). PP did not .!!... between -156 C
variant carriers and wild-type homozygotes (53 + 11 vs.
51+ 10; P= 0.22). Because of the high degree of linkage
i;, .1,.;I;,i ... between the 398 G>A and -156 G>C


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SNPs, blood pressure differences were similar when
compared by 398 G > A genotypes. For example, SBP
was 130 + 16 and 125 + 14 mmHg in 398A variant car-
riers and 398 G/G homozygotes, respectively (P= 0.033);
DBP was 78 11 and 74 11 mmHg, respectively
(P= 0.038); and PP was not different between groups
(53 + 11 vs. 51 + 10 mmHg in 398A carriers and 398 G/G
homozygotes, respectively; P= 0.362).
Age (P<0.001), sex (P< 0.008), and BMI (P< 0.002)
were common univariate predictors of SBP, DBP, and
PP. Furthermore, WBC count (P=0.10 for SBP;
P=0.076 for DBP) and both CXCL5 polymorphisms
(range P= 0.008 to 0.038) were additional predictors of
SBP and DBP, while smoking status was associated with
SBP alone (P= 0.038). In terms of circulating CRP and
ENA-78 levels, both biomarkers were significant for SBP
(P= 0.005 for CRP and P= 0.033 for ENA-78) and PP
(P = 0.001 for CRP and P= 0.007 for ENA-78) in univari-
ate analyses. Consistent with our previous report,
CXCLS genotype was associated with ENA-78 protein
concentrations in the plasma whereby variant carriers
at either SNP locus had higher protein concentrations
than wild-type homozygotes (P = 0.003; Figure 1).
In multivariate analysis of SBP, age, sex, BMI, and the
CXCLS -156 G > C promoter polymorphism were identi-
fied as significant variables (Table 2). The overall model
that included these variables explained 32.5% of the vari-
ability in SBP (P < 0.001). Consideration of the 398 G > A
polymorphism rather than the -156 G>C promoter
SNP resulted in a model in which only age, sex, and
BMI were significantly associated with SBP (R2 = 0.301;
P< 0.001).


N= 124 47
G/G C Carrier
CXCL5 -156G>C Genotype
Figure 1 Plasma ENA-78 by CXCL5 -156 G > C genotype.
P 0.003; data were similar for the exon 2 SNP, data not show
(P=0.001).


Table 2 Multivariate predictors of systolic blood pressure
in cardiovascular disease-free individuals


Variable

Constant
Age
Sex


0.313
-984


Standard
error
4.86
0.094


P value

<0.0001
0.001
<00001


BMI 0.637 0.160 <0.0001
-156 C carrier 4.93 2.30 0.034
R2=0.325; P<0.0001.


Age, sex, and the -156 G > C SNP were further asso-
ciated with DBP, along with WBC (Table 3). Consider-
ation of this promoter SNP (model R2 = 0.168; P< 0.0001)
was slightly more informative than consideration of the
398 G > A SNP (P= 0.067) in which case age (P <0.0001),
sex (P= 0.001), and WBC (P= 0.02) still remained signifi-
cant (model R2 = 0.145; P< 0.0001). In multivariable mod-
els of PP, only sex (P<0.004) and BMI (P<0.0001) were
significant (model R =0.247; P <0.0001).

Allelic expression imbalance
Allele-specific mRNA quantification was performed to
determine whether there is a functional basis for the dif-
ferences seen in blood pressure based on CXCLS geno-
types (see 'Materials and methods' section for rationale
of 398 G>A as marker SNP). Importantly, there was
consistently higher expression of CXCLS mRNA from
the 398A allele compared to the 398 G allele in hetero-
zygous individuals (Figure 2A). For example, individual
heterozygotes displayed anywhere from 2.2-fold to 3.4-
fold higher expression of 398A variant transcripts com-
pared to the 398 G allele, with a mean ratio of 2.9
(Figure 2B; P= 7.4E-15).

Discussion
Accumulating evidence points to a relationship between
inflammation and blood pressure. Data suggest that
WBC counts are associated with incident hyperten-
sion and correlated with blood pressure concentrations.
We hypothesized that WBC count is a surrogate for


Table 3 Multivariate predictors of diastolic blood
pressure in cardiovascular disease-free individuals


Variable

Constant
Aae


Standard
error
342
0063


-156 C carrier
WBC
R2 =0.168; P< 0.0001.


0.768


P value

<0.0001
<00001


0.374


0.041


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Patient ID


DNA
I mRNA


B
4.5
4-
3.5
3
398A:G 2
2.5
Allele
Ratio 2


Page 5 of 8


P=7.3877E-15


UI I
DNA mRNA
Figure 2 Allele-specific CXCL5 mRNA expression in leukocytes. (A) Allelic mRNA and DNA ratios were measured in 18 cardiovascular
disease-free individuals heterozygous for the 398 G >A SNP. The A/G ratios in DNA were close to 1 suggesting equal abundance of both alleles,
whereas there was consistently higher expression of mRNA from the 398A allele compared to the 398 G allele. (B) Pooled 398A/G ratios from 18
heterozygous individuals. The sample displayed 2.9-fold higher expression of 398A variant transcripts compared to the 398 G allele (P 7.4E-15).
Data are presented as mean+SD.


leukocytic chemokine activity and that the CXCL5 gene,
which encodes the neutrophil attractor ENA-78, may be
an important determinant of blood pressure. We demon-
strated a significant, independent relationship between
CXCL5 polymorphisms and SBP and DBP in the overall
population of CVD-free individuals. Variant carriers of the
-156 G > C promoter SNP had 7-mmHg and 4-mmHg
higher SBP and DBP, respectively, than those with the
wild-type -156 G/G genotype. Because of the epidemio-
logically significant difference in CVD risk conferred by


blood pressure differences of this magnitude, and since
variant carriers represent approximately 30% of the popu-
lation studied, CXCL5 polymorphisms should be consid-
ered as a potential novel biomarker of pre-hypertension,
hypertension, and CVD risk requiring future study. How-
ever, it is important to emphasize that genetic associa-
tions are preliminary and will require confirmation in
additional populations.
Of particular interest, WBC count (along with trad-
itional variables such as age, sex, smoking status, and







Beitelshees et al Human Genomics 2012, 6:9
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BMI) was significantly associated with SBP and DBP
in univariate analysis among CVD-free individuals. This
finding supports the report by Orakzai et al. that
demonstrated a relationship between WBC counts and
SBP among nearly 3,500 white individuals without CVD
and with SBP < 140 mmHg on entry [20]. It also supports
data from other clinical cohorts showing an associa-
tion between WBC count, major WBC components (e.g.,
!-!.....'.l:.!.l'i.,. and blood pressure '_i 1i i i] However,
in our analysis WBC count was no longer a significant
predictor of SBP when CXCLS genotype was included in
multivariable analysis, .1.. -:.. 1-, genotype may capture
the contribution of inflammation to SBP more effectively
than WBC count. WBC did, however, remain a signifi-
cant predictor of DBP in multivariate analysis, along with
age, sex, and CXCLS -156 G > C genotype.
To determine whether there is any functional basis for
an observed association between CXCLS variant alleles
and blood pressure, we performed allele expression im-
balance experiments in a subset of participants. The exo-
nic 398 G >A allele was chosen as the genetic marker
given its location in the coding region of the mRNA.
However, the 398 G/A heterozygous .(. ..i i i. (N= 18)
were also heterozygous for the promoter polymorphism,
which minimizes .. "i,'.i"..." of an association by dif-
fering genotypes at the upstream locus. It was noted that
variant carriers :i' .ii nearly threefold higher expres-
sion of variant CXCL5 mRNA transcripts from the 398A
allele. This novel '-.....,._ is consistent with our previous
observation that variant carriers exhibited higher plasma
and leukocyte-produced ENA-78 than ii I iype horno-
zygotes and that the promoter and exonic SNPs occur in
transcription factor I,-,.i;.'_ and .''"; ;.. enhancer sites,
respectively [33]. Given that the -156 G>C and 398
G >A SNPs are in near perfect linkage disequilibrium, it
is unclear which polymorphism is the causal variant and
functionally contributes to the blood pressure pheno-
type. However, the -156 G > C promoter SNP was more
significantly correlated with blood pressure in our study.
Further functional studies of these SNPs are warranted.
In addition to genotype and traditional covariates, we
included plasma CRP and ENA-78 protein concentra-
tions in our analyses. While CRP and ENA-78 were sig-
nificantly associated with SBP (and PP) in univariate
analyses, they fell out of the models when CXCL5 geno-
type was included. This ..* i that in our analyses,
genotype is more ..... .ti mai associated with the blood
pressure phenotype than systemically circulating con-
centrations of the ...-... :1. inflammatory mediator
CRP and the CXCL5 protein product ENA-78. While
this observation may appear somewhat contradictory, it
can be postulated that CXCL5 gene polymorphisms may
be better indicators of chemokine activity at the target
organ (e.g., endothelium) level than a measurement in the


circulation. Because of trans-acting influences on
systemic biomarker expression, polymorphisms in CXCLS
may be more robustly associated with blood pressure.
In fact, we have shown a similar finding in a different
population for the endothelial nitric oxide synthase gene
where NOS3 gene polymorphisms, but not measures of
circulating NO activity, were associated with arterial
stiffness in children with type 1 diabetes [42,43]. Further
support for this observation can be found in a case-
control study of the role of ENA-78 in patients with
ischemic stroke. Zaremba et al. demonstrated that
serum ENA-78 protein concentrations were not differ-
ent between stroke patients and controls; ..,a "'' .:i it
was demonstrated that ENA-78 concentrations were sig-
nificantly higher (twofold) in the cerebrospinal fluid of
stroke patients compared with controls [44]. Taken in
sum, it is possible that genotype more effectively cap-
tures the likelihood for local preponderance of chemo-
kine activity than plasma protein level.
In general, there is biological plausibility for the role of
CXCLS in CVD. For example, the protein product of
CXCL5, ENA-78, belongs to the same class of chemo-
kines as IL-8, IP-10, and I-TAC, which have been previ-
ously implicated in atherosclerotic inflammation [23,45].
ENA-78 has been shown to be chemotactic for neu-
trophils and stimulate .' ) -,..i,.i,. degranulation caus-
ing release of myeloperoxidase and generating reactive
oxygen species [24,25]. In addition, ENA-78 is involved
in platelet-dependent activation of monocytes, displays
angiogenic properties, and has been implicated in diseases
such as obesity, ii:, I t subclinical atherosclerosis, acute
coronary syndromes, ischemic stroke, abdominal aortic
aneurysm, and thrombosis [27-. '- I. 1i-51]. Hyperten-
sion is a risk factor for adverse events such as atheroscler-
osis, stroke, and abdominal aortic aneurysm, and ENA-78
is overexpressed in these situations. We have shown
CXCL5 polymorphisms to be associated with ENA-78
concentrations, blood pressure, and prognosis :;.i.,..
acute coronary syndromes [27,33]. Thus, the role of
CXCL5 in CVD should be further explored. As final
hypothesis-generating evidence of a link between the
CXCL5 pathway and blood pressure, stations have been
hypothesized to have mild ,'lI lli ii. -'. effects, and
we have shown that atorvastatin reduces ENA-78 produc-
tion from human endothelial cells in a dose-dependent
fashion [52,53]. Our findings, along with existing data,
support the need for future investigation of CXCL5 as a
hypertension- and CVD-susceptibility gene.

Competing interests
The authors declare that they have no competing interestss

Authors' contributions
ALB performed statistical analyses and drafted the manuscript CLA enrolled
the study su tjects and drafted t e manuscr pt HA assisted ,n the molecular
genetic studies and provided critical revision of the m 'anuscript TYL assisted


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in the mo ecular genetic studies GJW assisted in the molecular genetic
studies RSS assisted with the clinical study IZ conceived the manuscript,
enrolled the study subjects, and drafted the manuscript All authors read and
approved the final manuscilpti

Acknowledgments
We thank Dr Julle A Johnson for her thoughtful comments regarding the
manuscript We thank Lauren Burt and Lynda Stau'et "or their laboratory
assistance This work was supported by American Heart Association Florida/
Puerto Rico Aff late Scientist Development Grant 0435278B, American
College of Clinica Pharmacy Kos Dys lpidemia Research and
Phairmacotheiapy New Investigator Avvlds, American Associalhn of
Colleges of Pharmacy New Investigator Program Award, the Un:vers ty of
Colorado Denver General Clinica Research Center (RR00051), and N i C06
Grant RR17568 ALB is supported by K23 HLO91120

Author details
Division of Endocrinology, Diabetes and Nutrition, University of Maryland
School of Medicine, 660 W Redwood St, HH469, Baltimore, MD 21201, USA
2Department of Pharmaceutical Sciences, University of Colorado Skaggs
School of Pharmacy and Pharmaceutical Sciences, Aurora, CO 80045, USA
Department of Preventive Medicine and Institute for Genetic Medicine, Keck
School of Medicine, University of Southern California, Los Angeles, CA 90089,
USA 4Department of Pharmacotherapy and Translational Research, Center
for Pharmacogenomics, University of Florida College of Pharmacy,
Gainesville, FL 32610, USA SDivision of Cardiovascular Medicine and
Department of Veterans Affairs Medical Center, University of Florida College
of Medicine, Gainesville, FL 32603, USA

Received: 18 May 2012 Accepted: 18 May 2012
Published: 2 August 2012

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doi:1 0.1186/1479-7364-6-9
Cite this article as: Beltelshees et al CXCL5 polymorphisms are
associated with variable blood pressure in cardiovascular
disease-free adults. Human Genomics 2012 69


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PRIMARYRESEARCHOpenAccessCXCL5polymorphismsareassociatedwith variablebloodpressureincardiovascular disease-freeadultsAmberLBeitelshees1* †,ChristinaLAquilante2 †,HoomanAllayee3,TaimourYLangaee4,GregoryJWelder4, RichardSSchofield5andIssamZineh4AbstractObjective: Leukocytecounthasbeenassociatedwithbloodpressure,hypertension,andhypertensive complications.Wehypothesizedthatpolymorphismsinthe CXCL5 gene,whichencodestheneutrophilic chemokineENA-78,areassociatedwithbloodpressureincardiovasculardisease(CVD)-freeadultsandthat thesepolymorphismsarefunctional. Methodsandresults: Atotalof192community-dwellingparticipantswithoutCVDorriskequivalentswere enrolled.Two CXCL5 polymorphisms( 156G>C(rs352046)and398G>A(rs425535))weretestedforassociations withbloodpressure.Allele-specificmRNAexpressioninleukocyteswasalsomeasuredtodeterminewhether heterozygositywasassociatedwithallelicexpressionimbalance.In 156Cvariantcarriers,systolicbloodpressure (SBP)was7mmHghigherthanin 156G/Gwild-typehomozygotes(13117vs.12414mmHg; P =0.008). Similarly,diastolicbloodpressure(DBP)was4mmHghigherin 156Cvariantcarriers(7811vs.7411mmHg; P =0.013).InmultivariateanalysisofSBP,age,sex,bodymassindex,andthe 156G>Cpolymorphismwere identifiedassignificantvariables.Age,sex,andthe 156G>CSNPwerefurtherassociatedwithDBP,alongwith whitebloodcells.AllelicexpressionimbalanceandsignificantlyhighercirculatingENA-78concentrationswere notedforvariantcarriers. Conclusion: CXCL5 genepolymorphismsarefunctionalandassociatedwithvariablebloodpressureinCVD-free individuals.Theroleof CXCL5 asahypertension-andCVD-susceptibilitygeneshouldbefurtherexplored. Keywords: CXCL5,ENA-78,Bloodpressure,Hypertension,LeukocytesIntroductionTherelationshipbetweeninflammationandelevatedblood pressureisincreasinglybeingevaluated[1,2].Ithasbeen shownthatelevatedconcentrationsofprototypicalproinflammatorymarkerssuchasinterleukin-6,C-reactive protein(CRP),andtumornecrosisfactor-alphaare associatedwithincreasedbloodpressure,incidenceof hypertension,andthelikelihoodforhypertensivecomplications[3-14].Ithasbeenfurthersuggestedthat thisinflammatory-hypertensiverelationshipresultsfrom increasednumberoractivityofcommoncellular mediatorssuchaswhitebloodcells(WBC)[15,16].For example,studieshavedemonstratedelevatedWBCcount tobeassociatedwithincreasedincidenthypertensionas wellasincreasedbloodpressurewithinthenormaltoprehypertensiverange[17-22]. Althoughtheexactmechanisticrelationshipbetween leukocytosisandelevatedbloodpressureisunknown, itisplausiblethatlow-gradeinflammationmaybea contributingfactor.Inthisregard,WBCcountmaybea surrogatemarkerforincreasedactivationofinflammatory pathwaysthatcauseleukocyterecruitmentandactivation.Assuch,increasedactivityofleukocyticchemokines couldberelatedtoincreasedbloodpressure. Epithelialneutrophilactivator-78(ENA-78),akey leukocyticchemokinethatisbothaneutrophilattractor *Correspondence: abeitels@medicine.umaryland.edu†Equalcontributors1DivisionofEndocrinology,DiabetesandNutrition,UniversityofMaryland SchoolofMedicine,660W.RedwoodSt,HH469,Baltimore,MD21201,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Beitelsheesetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Beitelshees etal.HumanGenomics 2012, 6 :9 http://www.humgenomics.com/content/6/1/9

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andactivator,hasbeenimplicatedinmanydiseaseswith aninflammatorycomponent(e.g.,obesity,diabetes,subclinicalatherosclerosis,acutecoronarysyndromes)[23-32]. Wehavepreviouslyreportedthattwosinglenucleotide polymorphisms(SNPs),-156G>C(rs352046)and398 G>A(rs425535),inthegeneencodingENA-78( CXCL5) occurinsitesimportantfortranscriptionandexonsplicing[33].Inourpreviouswork,arelationshipexisted betweentheseSNPsandbothplasmaconcentrationsand leukocyteproductionoftheENA-78chemokineprotein [33].Wethenwentontoshowanassociationbetween the CXCL5 -156G>Cpolymorphismandworseoutcomesinpatientswithacutecoronarysyndromes[27]. Inthepresentwork,totheextentthatENA-78is importantinneutrophilrecruitmentanddegranulation, wehypothesizedthatoneorbothofthesepolymorphisms( 156G>Cand398G>A)couldbeassociated withdifferencesinbloodpressureinindividualswithout establishedcardiovasculardisease(CVD).Specifically, wehypothesizedthatrelativelyyoungindividualswithout knownCVDwhowerecarriersof CXCL5 variantalleles wouldexhibithighersystolicbloodpressure(SBP),diastolicbloodpressure(DBP),orpulsepressure(PP)than wild-typehomozygotes.Furthermore,toassesswhether therewasafunctionalroleforthesepolymorphisms, wemeasuredallele-specificmRNAexpressionof CXCL5 inleukocytesobtainedfromCVD-freeindividualswho wereheterozygousfortheSNPsatbothloci.MaterialsandmethodsStudypopulationThestudypopulationhasbeenpreviouslydescribed [33].Briefly,participantswererecruitedfromtwosites intheUSAandhadtobeatleast18yearsofagewithoutknownCVDorCVD-riskequivalents(e.g.,diabetes, peripheralvasculardisease,10-yearFraminghamRisk 20%)asdefinedbyNationalCholesterolEducationProgramcriteria[34].Otherexclusionswerepregnancy, malignancy,substanceabuse,androutineuseofmedicationsknowntoaffectWBCcountssuchassystemic steroidsandotheranti-inflammatoryagents.Individuals wereexcludedfromanalysisiftheyweretakingantihypertensivemedicationsforeithercardiovascularor non-cardiovascularindications(e.g.,migraine).Forblood pressuremeasurement,subjectswereseatedforatleast 5mininaquiet,temperature-controlledGeneralClinicalResearchCenter(GCRC)outpatientclinicroom, andtwobloodpressuremeasurementsweretakenat least5minapart.Theaverageoftheduplicateblood pressuremeasurementswasusedforthisinvestigation. Bloodsampleswereobtainedfromparticipantsenrolled inUniversityofFlorida-andColoradoMultipleInstitutionalReviewBoard(IRB)-approvedstudies.Allsubjects providedwritteninformedconsenttospecimenanddata useingeneticassociationandrelatedstudies.GenotypeandinflammatorybiomarkerdeterminationGenomicDNAwasisolatedfromwholebloodorbuccal cellsusingpreviouslydescribedmethods[35]. CXCL5 genotypesweredeterminedbypolymerasechainreaction(PCR)andpyrosequencing(Qiagen,Valencia,CA, USA)aswehavepreviouslydescribed[36].Circulating high-sensitivityCRP(asanon-specificmarkerofinflammation)wasmeasuredbytheShandsHospitalLaboratoryattheUniversityofFloridaandUniversity ofColoradoGCRC.ENA-78concentrationsweremeasuredbycytometricfluorescencedetectionaspreviously described(Luminex ™ 100ISsystem;LuminexCorp., Austin,TX,USA;FluorokineWMAPMultiplexHuman CytokinePanelA;R&DSystems,Minneapolis,MN, USA)[37].Sampleswerestoredat 80CuntilCRPand ENA-78detectionwasperformed.Allele-specificmRNAquantificationTodeterminewhethervariantcarrierstatusresultsin functionalchangesatthetranscriptionallevel,wequantifiedallele-specificmRNAtranscriptsfromleukocytes usingpyrosequencing-basedmethodology[38,39].Specifically,thepresenceorabsenceofallelicexpression imbalancewasdeterminedusingleukocytesobtained from18individualswhowereheterozygoticforboth the 156G>Cand398G>Apolymorphisms.The 398G>ASNPwaschosenasthegeneticbiomarkerin theseexperimentsbecauseitislocatedinthecodingregionof CXCL5 ,while 156G>Cisapromoterpolymorphismandassuchcannotbequantifiedatthe mRNAlevel.Becauseofthenearcompletelinkageof thestudiedSNPs,wechoseindividualswhowereheterozygotesatbothlocisothat398G>Agenotypemight serveasafunctionalsurrogatefortheupstreampromoterlocus. LeukocytemRNAwaspreparedfromapproximately 6106cellsfromeachindividualusingtheRNeasymini kit(Qiagen,Valencia,CA,USA).Cellswererinsed,lysed, andhomogenizedinbufferedsolutionsandsubsequently passedthroughtheRNeasyminicolumn(Qiagen, Valencia,CA,USA).Followingaseriesofwashesat roomtemperatureand15-minincubationwithDNase, concentrationsweredeterminedbyspectrophotometry (NanoDropTechnologies,Wilmington,DE,USA). cDNAwassynthesizedusingapproximately450ngof cellularRNAfromeachindividualusingaHighCapacitycDNAArchiveKit(AppliedBiosystems,Foster City,CA,USA)perprotocol.Conditionsforreverse transcriptionwere25Cfor10minfollowedby37Cfor 2h.cDNAqualitywasassessedbycomparingcDNA andDNAPCRproductsgeneratedusingintron-Beitelshees etal.HumanGenomics 2012, 6 :9 Page2of8 http://www.humgenomics.com/content/6/1/9

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spanningprimersbygelelectrophoresis.Forallelespecifictranscriptquantification,subjectDNAand cDNAunderwentPCRsimultaneouslyusingpreviously describedconditions[36].PCRproductsobtainedfor genotypedetermination(DNA)andtranscriptquantification(mRNA)wereassayedinparallelpyrosequencing reactionstominimizecyclevariability.Pyrosequencing analyseswereperformedinduplicateonthreeseparate PCRamplificationproducts,andtheresultswerepooled foranalysis.Peakheightsweredeterminedbythepyrosequencingallelequantificationalgorithm.Ingenomic DNA,theratioof398A:GallelesforDNAinheterozygotesisexpectedtobeapproximately1,whereassignificantdeviationsfromthisratioinmRNAwouldsuggest alleleexpressionimbalanceassociatedwiththevariant allele.StatisticalanalysesGenotypefrequenciesweredeterminedbyallelecounting,anddeparturesfromHardy-Weinbergequilibrium wereassessedbychi-squareanalyses.Differencesin bloodpressurebygenotypegroups(0,homozygousfor commonallele;1,heterozygousorhomozygousforvariantallele)werecomparedusingone-wayANOVA. Basedonthepreexistingsamplesizeandprevalenceof variantalleles,wehad80%powerwithatwo-sided of 0.05todetecta6-mmHgdifferenceinSBP,4-mmHg differenceinDBP,and4-mmHgdifferenceinPPbetweengenotypegroups.Multipleregressionanalysiswas performedifbloodpressuredifferenceswereseenacross genotypegroups.Covariatesformultipleregressionwere chosenthroughunivariateanalysesofage,sex,smoking status(0,non-smoker;1,currentsmoker),bodymass index(BMI),CRPconcentration,ENA-78concentration, andWBCcount.Anyvariablewitha P 0.1onunivariateanalysiswasenteredintothemultivariablemodel. Becauseofsmallnumbersofindividualswithinracial groups,analysescouldnotbeperformedwithinracial strata.However,race(0,white;1,non-white)was includedinallmultivariableanalyses,andarace-bygenotypeinteractiontermwasconsideredintheregressionmodelstoavoidspuriousassociationssecondaryto racialdifferencesinallelefrequency.Multipleregression usingstep-typeselectionmethodswasperformedto determinethejointeffectsof CXCL5 genotypesandclinicalvariablesonSBP,DBP,orPP.Allstatisticalanalyses wereperformedusingSPSS(version11.5,SPSSInc., Chicago,IL,USA)orSAS(version9.1,SASInstitute Inc.,Cary,NC,USA).A P value<0.05wasconsidered statisticallysignificant.ResultsBaselinedemographiccharacteristicsareshownin Table1.Participantswereonaverage3912yearsold withbloodpressuresof126/7515/11mmHg.-156 G>Cand398G>Agenotypesweredeterminedfor189 and188ofthe192individuals,respectively.Theoverall 156Cand398Aminorallelefrequencieswereboth 15%.Variantallelefrequenciesdifferedbyracewhereby the 156Callelefrequencywas14%,45%,and11%, and398Aallelefrequencywas13%,46%,and9%in Caucasians,blacks,andnon-blackHispanics,respectively.GenotypedistributionssatisfiedcriteriaforHardyWeinbergequilibrium(datanotshown).ThetwoSNPs wereinahighdegreeoflinkagedisequilibriumwith r2forCaucasian,black,andHispanicindividualsof0.82, 1.0,and0.51,respectively,inourstudypopulation.GenotypeassociationwithbloodpressureIn 156Cvariantcarriers,SBPwas7-mmHghigher thanin 156G/Gwild-typehomozygotes(13117vs. 12414mmHg; P =0.008).Similarly,DBPwas4-mmHg higherin 156Cvariantcarriers(7811vs.7411 mmHg; P =0.013).PPdidnotdifferbetween 156C variantcarriersandwild-typehomozygotes(5311vs. 5110; P =0.22).Becauseofthehighdegreeoflinkage disequilibriumbetweenthe398G>Aand 156G>C Table1BaselinecharacteristicsCharacteristic N =192 Age(meanSD,years)3912 Women(number(%))124(65) Race/ethnicity(number(%)) White148(77) Black12(6) Hispanic19(10) Other13(7) Familyheartdiseasehistory(number(%))29(15.1) Smoking(number(%))35(18) Bodymassindex(meanSD,kg/m2)29.67 Bloodpressure(meanSD,mmHg) Systolic12615 Diastolic7511 Pulsepressure(meanSD,mmHg)5110 Cholesterol(meanSD,mg/dLa) Total20143 LDL11836 HDL5517 Triglycerides139107 Whitebloodcellcount,(meanSD,109cells/L)6.32.0 C-reactiveprotein(median(range),mg/Lb)1.78(0.1 – 16.9) ENA-78(median(range),pg/mLb)362(32.2 – 3970)aTotal,HDL,andtriglyceridesavailablein94%ofsubjects;LDLavailablein 92%ofsubjects.bCRPandENA-78availablefor88%and91%ofsubjects, respectively.Beitelshees etal.HumanGenomics 2012, 6 :9 Page3of8 http://www.humgenomics.com/content/6/1/9

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SNPs,bloodpressuredifferencesweresimilarwhen comparedby398G>Agenotypes.Forexample,SBP was13016and12514mmHgin398Avariantcarriersand398G/Ghomozygotes,respectively( P =0.033); DBPwas7811and7411mmHg,respectively ( P =0.038);andPPwasnotdifferentbetweengroups (5311vs.5110mmHgin398Acarriersand398G/G homozygotes,respectively; P =0.362). Age( P 0.001),sex( P 0.008),andBMI( P 0.002) werecommonunivariatepredictorsofSBP,DBP,and PP.Furthermore,WBCcount( P =0.10forSBP; P =0.076forDBP)andboth CXCL5 polymorphisms (range P =0.008to0.038)wereadditionalpredictorsof SBPandDBP,whilesmokingstatuswasassociatedwith SBPalone( P =0.038).IntermsofcirculatingCRPand ENA-78levels,bothbiomarkersweresignificantforSBP ( P =0.005forCRPand P =0.033forENA-78)andPP ( P =0.001forCRPand P =0.007forENA-78)inunivariateanalyses.Consistentwithourpreviousreport, CXCL5 genotypewasassociatedwithENA-78protein concentrationsintheplasmawherebyvariantcarriers ateitherSNPlocushadhigherproteinconcentrations thanwild-typehomozygotes( P =0.003;Figure1). InmultivariateanalysisofSBP,age,sex,BMI,andthe CXCL5 -156G>Cpromoterpolymorphismwereidentifiedassignificantvariables(Table2).Theoverallmodel thatincludedthesevariablesexplained32.5%ofthevariabilityinSBP( P <0.001).Considerationofthe398G>A polymorphismratherthanthe 156G>Cpromoter SNPresultedinamodelinwhichonlyage,sex,and BMIweresignificantlyassociatedwithSBP( R2=0.301; P <0.001). Age,sex,andthe 156G>CSNPwerefurtherassociatedwithDBP,alongwithWBC(Table3).ConsiderationofthispromoterSNP(model R2=0.168; P <0.0001) wasslightlymoreinformativethanconsiderationofthe 398G>ASNP( P =0.067)inwhichcaseage( P <0.0001), sex( P =0.001),andWBC( P =0.02)stillremainedsignificant(model R2=0.145; P <0.0001).InmultivariablemodelsofPP,onlysex( P <0.004)andBMI( P <0.0001)were significant(model R2=0.247; P <0.0001).AllelicexpressionimbalanceAllele-specificmRNAquantificationwasperformedto determinewhetherthereisafunctionalbasisforthedifferencesseeninbloodpressurebasedon CXCL5 genotypes(see ‘ Materialsandmethods ’ sectionforrationale of398G>AasmarkerSNP).Importantly,therewas consistentlyhigherexpressionof CXCL5 mRNAfrom the398Aallelecomparedtothe398Galleleinheterozygousindividuals(Figure2A).Forexample,individual heterozygotesdisplayedanywherefrom2.2-foldto3.4foldhigherexpressionof398Avarianttranscriptscomparedtothe398Gallele,withameanratioof2.9 (Figure2B; P =7.4E-15).DiscussionAccumulatingevidencepointstoarelationshipbetween inflammationandbloodpressure.Datasuggestthat WBCcountsareassociatedwithincidenthypertensionandcorrelatedwithbloodpressureconcentrations. WehypothesizedthatWBCcountisasurrogatefor 47 124 N =CXCL5 -156G>C GenotypeC Carrier G/G 2000 1500 1000 500 0 Figure1 PlasmaENA-78by CXCL5 -156G>Cgenotype. P =0.003;dataweresimilarfortheexon2SNP,datanotshown ( P =0.001). Table2Multivariatepredictorsofsystolicbloodpressure incardiovasculardisease-freeindividualsVariable Standard error P value Constant1004.86<0.0001 Age0.3130.0940.001 Sex 9.842.12<0.0001 BMI0.6370.160<0.0001 156Ccarrier4.932.300.034R2=0.325; P <0.0001. Table3Multivariatepredictorsofdiastolicblood pressureincardiovasculardisease-freeindividualsVariable Standard error P value Constant63.133.42<0.0001 Age0.2470.063<0.0001 Sex 5.8011.549<0.0001 156Ccarrier3.7351.6300.023 WBC0.7680.3740.041R2=0.168; P <0.0001.Beitelshees etal.HumanGenomics 2012, 6 :9 Page4of8 http://www.humgenomics.com/content/6/1/9

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leukocyticchemokineactivityandthatthe CXCL5 gene, whichencodestheneutrophilattractorENA-78,maybe animportantdeterminantofbloodpressure.Wedemonstratedasignificant,independentrelationshipbetween CXCL5 polymorphismsandSBPandDBPintheoverall populationofCVD-freeindivi duals.Variantcarriersofthe 156G>CpromoterSNPhad7-mmHgand4-mmHg higherSBPandDBP,respectively,thanthosewiththe wild-type 156G/Ggenotype.BecauseoftheepidemiologicallysignificantdifferenceinCVDriskconferredby bloodpressuredifferencesofthismagnitude,andsince variantcarriersrepresentapproximately30%ofthepopulationstudied, CXCL5 polymorphismsshouldbeconsideredasapotentialnovelbiomarkerofpre-hypertension, hypertension,andCVDriskrequiringfuturestudy.However,itisimportanttoemphasizethatgeneticassociationsarepreliminaryandwillrequireconfirmationin additionalpopulations. Ofparticularinterest,WBCcount(alongwithtraditionalvariablessuchasage,sex,smokingstatus,and Patient ID 123456789101112131415161718 398A:G Allelic Ratio 0 1 2 3 4 5 DNA mRNA A 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 DNAmRNA 398A:G Allele RatioP=7.3877E-15B Figure2 Allele-specific CXCL5 mRNAexpressioninleukocytes. ( A )AllelicmRNAandDNAratiosweremeasuredin18cardiovascular disease-freeindividualsheterozygousforthe398G>ASNP.TheA/GratiosinDNAwerecloseto1suggestingequalabundanceofbothalleles, whereastherewasconsistentlyhigherexpressionofmRNAfromthe398Aallelecomparedtothe398Gallele.( B )Pooled398A/Gratiosfrom18 heterozygousindividuals.Thesampledisplayed2.9-foldhigherexpressionof398Avarianttranscriptscomparedtothe398Gallele( P =7.4E-15). DataarepresentedasmeanSD. Beitelshees etal.HumanGenomics 2012, 6 :9 Page5of8 http://www.humgenomics.com/content/6/1/9

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BMI)wassignificantlyassociatedwithSBPandDBP inunivariateanalysisamongCVD-freeindividuals.This findingsupportsthereportbyOrakzaietal.that demonstratedarelationshipbetweenWBCcountsand SBPamongnearly3,500whiteindividualswithoutCVD andwithSBP<140mmHgonentry[20].Italsosupports datafromotherclinicalcohortsshowinganassociationbetweenWBCcount,majorWBCcomponents(e.g., neutrophils),andbloodpressure[21,22,40,41].However, inouranalysisWBCcountwasnolongerasignificant predictorofSBPwhen CXCL5 genotypewasincludedin multivariableanalysis,suggestinggenotypemaycapture thecontributionofinflammationtoSBPmoreeffectively thanWBCcount.WBCdid,however,remainasignificantpredictorofDBPinmultivariateanalysis,alongwith age,sex,and CXCL5 -156G>Cgenotype. Todeterminewhetherthereisanyfunctionalbasisfor anobservedassociationbetween CXCL5 variantalleles andbloodpressure,weperformedalleleexpressionimbalanceexperimentsinasubsetofparticipants.Theexonic398G>Aallelewaschosenasthegeneticmarker givenitslocationinthecodingregionofthemRNA. However,the398G/Aheterozygousindividuals( N =18) werealsoheterozygousforthepromoterpolymorphism, whichminimizesconfoundingofanassociationbydifferinggenotypesattheupstreamlocus.Itwasnotedthat variantcarriersdisplayednearlythreefoldhigherexpressionofvariant CXCL5 mRNAtranscriptsfromthe398A allele.Thisnovelfindingisconsistentwithourprevious observationthatvariantcarriersexhibitedhigherplasma andleukocyte-producedENA-78thanwild-typehomozygotesandthatthepromoterandexonicSNPsoccurin transcriptionfactorbindingandsplicingenhancersites, respectively[33].Giventhatthe 156G>Cand398 G>ASNPsareinnearperfectlinkagedisequilibrium,it isunclearwhichpolymorphismisthecausalvariantand functionallycontributestothebloodpressurephenotype.However,the 156G>CpromoterSNPwasmore significantlycorrelatedwithbloodpressureinourstudy. FurtherfunctionalstudiesoftheseSNPsarewarranted. Inadditiontogenotypeandtraditionalcovariates,we includedplasmaCRPandENA-78proteinconcentrationsinouranalyses.WhileCRPandENA-78weresignificantlyassociatedwithSBP(andPP)inunivariate analyses,theyfelloutofthemodelswhen CXCL5 genotypewasincluded.Thissuggeststhatinouranalyses, genotypeismoresignificantlyassociatedwiththeblood pressurephenotypethansystemicallycirculatingconcentrationsofthenon-specificinflammatorymediator CRPandthe CXCL5 proteinproductENA-78.While thisobservationmayappearsomewhatcontradictory,it canbepostulatedthat CXCL5 genepolymorphismsmay bebetterindicatorsofchemokineactivityatthetarget organ(e.g.,endothelium)levelthanameasurementinthe circulation.Becauseoftrans-actinginfluenceson systemicbiomarkerexpression,polymorphismsin CXCL5 maybemorerobustlyassociatedwithbloodpressure. Infact,wehaveshownasimilarfindinginadifferent populationfortheendothelialnitricoxidesynthasegene where NOS3 genepolymorphisms,butnotmeasuresof circulatingNOactivity,wereassociatedwitharterial stiffnessinchildrenwithtype1diabetes[42,43].Further supportforthisobservationcanbefoundinacase – controlstudyoftheroleofENA-78inpatientswith ischemicstroke.Zarembaetal.demonstratedthat serumENA-78proteinconcentrationswerenotdifferentbetweenstrokepatientsandcontrols;contrarily,it wasdemonstratedthatENA-78concentrationsweresignificantlyhigher(twofold)inthecerebrospinalfluidof strokepatientscomparedwithcontrols[44].Takenin sum,itispossiblethatgenotypemoreeffectivelycapturesthelikelihoodforlocalpreponderanceofchemokineactivitythanplasmaproteinlevel. Ingeneral,thereisbiologicalplausibilityfortheroleof CXCL5 inCVD.Forexample,theproteinproductof CXCL5 ,ENA-78,belongstothesameclassofchemokinesasIL-8,IP-10,andI-TAC,whichhavebeenpreviouslyimplicatedinatheroscleroticinflammation[23,45]. ENA-78hasbeenshowntobechemotacticforneutrophilsandstimulateneutrophilicdegranulationcausingreleaseofmyeloperoxidaseandgeneratingreactive oxygenspecies[24,25].Inaddition,ENA-78isinvolved inplatelet-dependentactivationofmonocytes,displays angiogenicproperties,andhasbeenimplicatedindiseases suchasobesity,diabetes,subclinicalatherosclerosis,acute coronarysyndromes,ischemicstroke,abdominalaortic aneurysm,andthrombosis[27,28,32,44,46-51].Hypertensionisariskfactorforadverseeventssuchasatherosclerosis,stroke,andabdominalaorticaneurysm,andENA-78 isoverexpressedinthesesituations.Wehaveshown CXCL5 polymorphismstobeassociatedwithENA-78 concentrations,bloodpressure,andprognosisfollowing acutecoronarysyndromes[27,33].Thus,theroleof CXCL5 inCVDshouldbefurtherexplored.Asfinal hypothesis-generatingevidenceofalinkbetweenthe CXCL5 pathwayandbloodpressure,statinshavebeen hypothesizedtohavemildantihypertensiveeffects,andwehaveshownthatatorvastatinreducesENA-78productionfromhumanendothelialcellsinadose-dependent fashion[52,53].Ourfindings,alongwithexistingdata, supporttheneedforfutureinvestigationof CXCL5 asa hypertension-andCVD-susceptibilitygene.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions ALBperformedstatisticalanalysesanddraftedthemanuscript.CLAenrolled thestudysubjectsanddraftedthemanuscript.HAassistedinthemolecular geneticstudiesandprovidedcriticalrevisionofthemanuscript.TYLassistedBeitelshees etal.HumanGenomics 2012, 6 :9 Page6of8 http://www.humgenomics.com/content/6/1/9

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inthemoleculargeneticstudies.GJWassistedinthemoleculargenetic studies.RSSassistedwiththeclinicalstudy.IZconceivedthemanuscript, enrolledthestudysubjects,anddraftedthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Acknowledgments WethankDr.JulieA.Johnsonforherthoughtfulcommentsregardingthe manuscript.WethankLaurenBurtandLyndaStaufferfortheirlaboratory assistance.ThisworkwassupportedbyAmericanHeartAssociationFlorida/ PuertoRicoAffiliateScientistDevelopmentGrant0435278B,American CollegeofClinicalPharmacyKosDyslipidemiaResearchand PharmacotherapyNewInvestigatorAwards,AmericanAssociationof CollegesofPharmacyNewInvestigatorProgramAward,theUniversityof ColoradoDenverGeneralClinicalResearchCenter(RR00051),andNIHC06 GrantRR17568.ALBissupportedbyK23HL091120. Authordetails1DivisionofEndocrinology,DiabetesandNutrition,UniversityofMaryland SchoolofMedicine,660W.RedwoodSt,HH469,Baltimore,MD21201,USA.2DepartmentofPharmaceuticalSciences,UniversityofColoradoSkaggs SchoolofPharmacyandPharmaceuticalSciences,Aurora,CO80045,USA.3DepartmentofPreventiveMedicineandInstituteforGeneticMedicine,Keck SchoolofMedicine,UniversityofSouthernCalifornia,LosAngeles,CA90089, USA.4DepartmentofPharmacotherapyandTranslationalResearch,Center forPharmacogenomics,UniversityofFloridaCollegeofPharmacy, Gainesville,FL32610,USA.5DivisionofCardiovascularMedicineand DepartmentofVeteransAffairsMedicalCenter,UniversityofFloridaCollege ofMedicine,Gainesville,FL32603,USA. Received:18May2012Accepted:18May2012 Published:2August2012 References1.WatsonT,GoonPK,LipGY: Endothelialprogenitorcells,endothelial dysfunction,inflammation,andoxidativestressinhypertension. 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JClinEndocrinolMetab 2010, 95: 3926 – 3932. 31.KeeleyEC,MoormanJR,LiuL,GimpleLW,LipsonLC,RagostaM, TaylorAM,LakeDE,BurdickMD,MehradB,StrieterRM: Plasmachemokine levelsareassociatedwiththepresenceandextentofangiographic coronarycollateralsinchronicischemicheartdisease. PLoSOne 2011, 6: e21174. 32.ChenL,YangZ,LuB,LiQ,YeZ,HeM,HuangY,WangX,ZhangZ, WenJ,LiuC,QuS,HuR: SerumCXCligand5isanewmarkerof subclinicalatherosclerosisintype2diabetes. ClinEndocrinol(Oxf)2011, 75 (6):766 – 770.Beitelshees etal.HumanGenomics 2012, 6 :9 Page7of8 http://www.humgenomics.com/content/6/1/9

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ui 1750-1326-7-46
ji 1750-1326
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dochead Research article
bibl
title
p Identification of BACE2 as an avid ß-amyloid-degrading protease
aug
au id A1 snm Abdul-Haymi Ofnm Samerinsr iid I1 email abdulhay.samer@mayo.edu
A2 SaharaTomokotomoko.sahara@mbi.ufl.edu
A3 McBrideMelindaleissring.malcolm@mayo.edu
A4 KangDongcheulkang.dongcheul@mayo.edu
A5 ca yes LeissringAMalcolmleissring@mayo.edu
insg
ins Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Birdsall Bldg., Rm. 117, Jacksonville, FL, 32224, USA
source Molecular Neurodegeneration
issn 1750-1326
pubdate 2012
volume 7
issue 1
fpage 46
url http://www.molecularneurodegeneration.com/content/7/1/46
xrefbib pubidlist pubid idtype doi 10.1186/1750-1326-7-46pmpid 22986058
history rec date day 16month 3year 2012acc 1682012pub 1792012
cpyrt 2012collab Abdul-Hay et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Amyloid-ß-protein
Alzheimer disease
ß-site APP-cleaving enzyme-1
ß-site APP-cleaving enzyme-2
Functional screen
Gene therapy
Protease
Proteolytic degradation
abs
sec
st
Abstract
Background
Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer’s disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays.
Results
The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds—Phe19-Phe20, Phe20-Ala21, and Leu34-Met35—with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE.
Conclusions
This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.
bdy
Background
Alzheimer disease (AD) is a progressive and presently incurable neurodegenerative disorder characterized by abnormal accumulation of the amyloid β-protein (Aβ) in brain regions important for mnemonic and cognitive functions. Aß is a heterogeneous mixture of peptides ranging from 37 to 43 amino acids in length abbrgrp
abbr bid B1 1
produced via sequential cleavage of the amyloid precursor protein (APP) by BACE1 and the presenilin/γ-secretase complex
B2 2
B3 3
B4 4
. Autosomal-dominant mutations in 3 genes—APP and presenilin-1 and −2—are known to cause rare, familial forms of AD either by increasing the production of all forms of Aß or by increasing the relative production of longer, more amyloidogenic forms, such as Aß42
B5 5
. Nevertheless, the precise mechanisms underlying sporadic AD, which makes up the vast majority of cases, remain to be elucidated.Aß-degrading proteases (AßDPs) are potent regulators of cerebral Aß levels and, as such, represent important players in the etiology and potential treatment of AD
B6 6
. Amyloidogenesis and downstream cytopathology can be attenuated and even completely prevented by enhancing the activity of any of several AßDPs, while, conversely, genetic deletion of one or more AßDPs leads to significant elevations in cerebral Aß
B7 7
. Significantly, patients with sporadic AD were recently shown to exhibit defects in the clearance of Aß (rather than increases in its production)
B8 8
and, in light of the large body of evidence implicating AßDPs in the regulation of cerebral Aß levels
7
, it is reasonable to infer that defects in one or more AßDPs could contribute to impaired Aß clearance. While more than twenty proteases are now known to degrade Aß
7
, these were not identified through any systematic approach, but instead emerged haphazardously from a disconnected set of largely serendipitous discoveries. Nevertheless, essentially all AßDPs now known to regulate Aß in vivo were originally identified through exclusively in vitro or cell-based approaches
B9 9
.To discover new AßDPs more systematically, we conducted an unbiased, cell-based, functional screen of 352 proteases in the human genome. The top Aß-lowering protease emerging from this screen was ß-site APP-cleaving enzyme-2 (BACE2)
B10 10
. Previous studies have shown that BACE2 can lower Aß levels via α-secretase-like cleavage of APP within the Aß sequence
B11 11
B12 12
B13 13
B14 14
B15 15
B16 16
, an activity that has been dubbed “θ-secretase”
B17 17
. However, we found that BACE2 is also a remarkably avid AßDP, with a catalytic efficiency exceeding all other known AßDPs except insulin-degrading enzyme (IDE).
Results and discussion
Functional screen for novel AßDPs
To identify novel AßDPs, we performed a cell-based functional screen using a commercial library consisting of 352 full-length, sequence-verified, human cDNAs encoding diverse members of all protease classes. We experimented with several approaches before settling on a final configuration for the primary screen. Assays designed to monitor degradation of exogenous Aß were found to be confounded by the highly dominant effect of IDE, which mediates the vast majority of extracellular Aß degradation in cultured cells
B18 18
B19 19
B20 20
. Transient transfection of cDNAs into cell lines stably expressing APP was also tried, but this approach suffered from incomplete transfection efficiency, which attenuated the effect on net extracellular Aß levels. We therefore elected to conduct the screen by co-transfecting protease-encoding cDNAs, together with positive and negative controls, into a rodent cell line (CHO cells) together with a plasmid encoding wild-type human APP fused to alkaline phosphatase (AP) (see Figure figr fid F1 1A; it Methods). Use of the APP-AP construct ensured that human Aβ production was limited to cells also expressing candidate AßDPs, while also providing an internal control for transfection efficiency (via AP activity). Importantly, the co-transfection strategy also increased the likelihood of detecting AßDPs that degrade Aß intracellularly, prior to its secretion, in addition to those that act exclusively extracellularly. Cytotoxicity was also quantified via an MTT conversion assay, but no significant cell death was detected so these data were not incorporated into subsequent analyses. The screen was performed in quadruplicate and, for each well, the ratio of Aß40 concentration to AP activity was calculated, then normalized to appropriate intra-plate controls (Figure 1B).
fig Figure 1caption Overview and outcome of functional screening for novel Aß-lowering proteasestext
b Overview and outcome of functional screening for novel Aß-lowering proteases. A, Cartoon illustrating the overall design of the screen. Briefly, an arrayed collection of 352 protease-encoding cDNAs was cotransfected into CHO cells together with an APP-AP fusion construct. Following a medium change and overnight incubation, Aß40 levels, AP activity, and cytotoxicity (via MTT assay) were analyized in the resulting conditioned media. B, Results of the screen, expressed as [Aß40]/AP ratios normalized to intraplate controls. Data are mean of 4 replicates. Note that the largest decrease in Aß levels by far was acheived by BACE2. C, Confirmation of the results of the screen. Following scale-up and sequence verification, cDNAs encoding BACE2 and its homolog, BACE1, were cotransfected together with APP-AP into CHO cells. Consistent with the outcome of the medium-throughput screen, BACE2, but not BACE1, expression resulted in significant decreases in the levels of both Aß40 and Aß42. Data are mean ± SEM of 4 replicates, and are normalized to vector-only controls.
graphic file 1750-1326-7-46-1 From among the 352 proteases examined, by far the largest decrease in normalized Aß levels (97 ± 1.2%) was induced by BACE2, which was in fact the only protease to lower Aß levels more than 75%, our pre-determined cut-off for viable hits (Figure 1B).
BACE2 transfection lowers Aβ levels
To confirm and extend the results obtained in the cDNA screen, we compared the degree to which overexpression of BACE2 and its homolog BACE1
B21 21
affected the net production of different Aß species. Consistent with the results of the primary screen, BACE2 transfection in CHO cells decreased the levels of both Aβ40 and Aβ42 (Figure 1C). Overexpression of BACE1 in this cell type, by contrast, had no effect on net Aß levels (Figure 1C). We note that BACE1 overexpression would not be expected to increase Aß production in CHO cells, since previous studies have established that γ-secretase, rather than ß-secretase, is the rate-limiting step in Aß production in this cell type
B22 22
.
BACE2 and BACE1 degrade Aß in vitro
Expression of BACE2 in cells could lower Aß levels either directly, via proteolytic degradation, or indirectly, via alternative mechanisms such as hydrolysis of APP or APP C-terminal fragments (CTFs)
11
12
13
14
15
16
. To distinguish these possibilities, we tested the ability of recombinant BACE2 to hydrolyze synthetic Aß in vitro, using a well-established fluorescence polarization-based Aß degradation assay
B23 23
. Recombinant BACE2 was found to avidly degrade Aß in this paradigm, confirming that BACE2 is indeed a bona fide AßDP (Figure F2 2A). Recombinant BACE1 also hydrolyzed Aß, indicating that it too is an AßDP (Figure 2B). However, BACE1 was much less efficient than BACE2, requiring 24 h to degrade Aß to a similar extent as was achieved following a 10-min incubation with BACE2 (Figure 2B). Based on these results, the efficiency of BACE1 would appear to be ~150-fold lower than that of BACE2.
Figure 2BACE2 degrades Aß in vitro
BACE2 degrades Aß in vitro. A, Percent Aß remaining following incubation with different concentrations of recombinant BACE2 for various lengths of time. Data are mean ± SEM of 4 replicates, normalized to protease-free controls. B, Comparison the relative Aß-degrading ability of recombinant BACE2 vs. BACE1. Note that 24 h incubation with BACE1 was required to achieve approximately the same extent of degradation as effected by BACE2 in 10 min. Data are mean ± SEM of 3 replicates, normalized to protease-free controls. C, BACE2 activity is pH dependent. Percent Aß degradation catalyzed by equivalent amounts of BACE2 at different pH values. Data are mean ± SEM of 4 replicates. D, BACE2 does not degrade fibrillar Aß. Lack of effect of BACE2 (10 nM) on preformed Aß42 fibrils following incubation at 37°C for 5 d, as determined by thioflavin T fluorescence. Data are mean ± SEM of 3 replicates.
1750-1326-7-46-2
BACE2-mediated Aß degradation is pH-dependent
As an aspartyl protease, the catalytic efficiency of BACE2 is expected to be pH-dependent. To confirm this, we compared the rate of hydrolysis of Aß40 across a range a pH values. Consistent with expectations, BACE2 was found to be maximally effective at pH 3.5 (Figure 2C), and decreasingly effective at higher pH values. These findings strongly suggest that BACE2 would not be operative at the cell surface or within the extracellular space.
BACE2 does not degrade fibrillar Aß
Individual AßDPs can be categorized in terms of their ability or inability to degrade fibrillar forms of Aß. Many well-established AßDPs, such as IDE and NEP, avidly degrade monomeric Aß but cannot degrade fibrillar forms and are therefore categorized as pure peptidases. Others, such as plasmin, degrade Aß fibrils and thus can also be categorized as fibrilases
7
. To determine to which category BACE2 belongs, we incubated recombinant BACE2 with pre-formed fibrils of Aß42 and quantified the degree of aggregation by thioflavin T fluorescence. No significant reduction in aggregation was observed, even following incubation at 37°C for up to 3 d (Figure 2D). These results suggest that, as is true for the majority of AßDPs
7
, BACE2 does not degrade Aß fibrils.
BACE2 cleaves Aß at 3 sites
We next investigated which peptide bond(s) within Aβ are hydrolyzed by BACE2 and BACE1. To that end, we co-incubated N-terminally biotinylated Aβ40 or Aβ42 (300nM) with BACE2 (5 nM) and analyzed the products by immunoprecipitation/mass spectrometry (IP/MS) (see Methods). Within 1 h, BACE2 almost completely hydrolyzed both Aß species, generating the shorter fragment, Aβ34, in both cases (Figure F3 3A-D). To test whether any additional cleavages can occur, we incubated N-terminally biotinylated Aβ40 (300 nM) with a larger amount of BACE2 (25 nM) for 1 and 24 h. At these higher concentrations and longer incubation times, Aβ19 and Aβ20 were the principal N-terminal fragments remaining at the end of the reaction (Figure 3E-F). Collectively, these in vitro results suggest that BACE2 cleaves Aβ at three different positions: Phe19-Phe20, Phe20-Ala21, and Leu34-Met35, with the latter cleavage site being the initial and principal one, as is consistent with previous observations
13
14
B24 24
.
Figure 3Determination of peptide bonds within Aß hydrolyzed by BACE2
Determination of peptide bonds within Aß hydrolyzed by BACE2. Top, Summary of cleavage sites determined from data in AF, showing the major site (block arrow) and two minor sites (arrow heads). At t = 0 (A, C), intact Aß42 (A) and Aß40 (C) represent the only species present. Following incubation of Aß42 and Aß40 with 5nM BACE2 for 1 h (BD, respectively), the full-length Aß species are essentially completely absent and replaced by Aß34. E,F, Additional Aß cleavage products are produced following incubation with larger amounts of BACE2 (25 nM) for longer lengths of time. By 1 h (E), a new peak corresponding to Aß20 is produced. By 24 h (F), Aß20 becomes the major species present, and Aß19 is also produced. Double-charged fragments are denoted by “+”, and “*” represents the modification of a fragment by AEBSF, which leads to a 183-Da increase in MW, as previously reported B46 46.
1750-1326-7-46-3 To confirm whether BACE2 cleaves Aβ at the same sites in a more physiological setting, we analyzed Aß species in the conditioned media of cells expressing APP-AP either alone or together with BACE2 by IP/MS (see Methods). As expected for cells expressing APP-AP alone, the medium from these cells contained Aβ42, Aβ40, Aβ39, Aβ38, and Aβ37 (Figure F4 4A). BACE2 expression suppressed the signal of all of these species, and new peaks corresponding to Aβ19, Aβ20, and Aβ34 emerged (Figure 4B), confirming that the cleavage sites mediated by BACE2 in vitro are also hydrolyzed in intact cells. The appearance of Aß34 is particularly notable, because cleavage at position 34 can only occur after production of full-length Aß, as this peptide bond is positioned within the transmembrane domain of APP, as has been shown previously
24
. Although this result clearly indicates that BACE2 does indeed degrade Aß after it is produced, it is not possible to quantify the extent to which the Aß19 and Aß20 peaks are the result of θ-secretase activity or subsequent degradation of the Aß34 fragment (or full-length Aß). As a consequence, it is difficult to estimate the exact extent to which the Aß-lowering effect of BACE2 can be assigned to non-amyloidogenic processing versus Aß degradation per se in experimental paradigms of this type.
Figure 4Overexpression of BACE2 in cells yields Aß fragments identical to those produced in vitro
Overexpression of BACE2 in cells yields Aß fragments identical to those produced in vitro. AB, Spectra of Aß fragments determined by IP/MS analysis of the conditioned media of CHO cells transfected with APP and empty vector (A) or APP and BACE2 (B) (see Methods).A, APP expression alone produces peaks corresponding to Aß42, Aß40, Aß39, Aß38 and Aß37. B, Co-expression of APP and BACE2 results in decreases in the relative abundance of the aforementioned Aß species and the appearance of three new fragments: Aß34, Aß20 and Aß19. Double-charged fragments are denoted by “+”, and “*” represents the modification of a fragment by AEBSF, which leads to a 183-Da increase in MW, as previously reported 46.
1750-1326-7-46-4
BACE2 degrades Aß more efficiently than well-established AßDPs
Having established BACE2 as an AßDP, we next investigated how BACE2 compares to other known AßDPs in terms the ability to degrade Aß in vitro and to lower net Aß levels in cells. To compare the relative efficiency of BACE2 in vitro, we monitored the degradation of a fixed amount of Aß (200 nM) by recombinant BACE2 (5 nM) as compared to equal quantities of several well-established AßDPs, including IDE, NEP and plasmin. Under these conditions, BACE2 hydrolyzed Aß more efficiently than all other AßDPs except IDE (Figure F5 5A). We note that the concentration of Aß used in this experiment was considerably lower than the K
sub M for each of the proteases tested (see
23
and below), making the initial velocity of this reaction a good index of the relative catalytic efficiency.
Figure 5Comparison of the efficacy of BACE2 relative to other well-established AßDPs in vitro and in cultured cells
Comparison of the efficacy of BACE2 relative to other well-established AßDPs in vitro and in cultured cells. A, Degradation of Aß in vitro by equivalent nominal concentrations (5 nM) of recombinant BACE2, IDE, NEP and plasmin. Note that BACE2 degrades Aß at a faster rate than NEP and plasmin, but not IDE. B,C, Effects on Aß40 (A) and Aß42 (C) levels following cotransfection of CHO cells with APP together with equivalent quantities of cDNAs encoding BACE2, ECE1b and IDE. In good agreement with the results in vitro (A), BACE2 lowers the levels of both Aß species to an extent exceeding NEP and ECE1b, but comparable to IDE. Data are mean ± SEM of 4 replications, normalized to controls cotransfected with empty vector (Vo).
1750-1326-7-46-5
Kinetics of Aß degradation by BACE2
To investigate the catalytic efficiency of BACE2 more quantititatively, we determined the kinetics of degradation of both Aß40 and Aß42 by BACE2 (see Methods). For this analysis, we were careful to use freshly prepared batches of monomeric human Aβ40 and Aβ42 peptides, which we routinely prepare by size-exclusion chromatography and which have been extensively characterized
B25 25
B26 26
. BACE2 cleaved both Aß species with similar kinetics, exhibiting apparent K
M values in the low micromolar range and albeit with apparent k
cat values slightly higher for Aß40 relative to Aß42 (0.135 ± 0.016 minsup -1 and 0.025 ± 0.005 min-1, respectively; Table tblr tid T1 1). In terms of catalytic efficiency (k
cat/K
M), BACE2 degrades Aβ40 approximately 4-fold more efficiently than Aβ42 (Table 1). These parameters exceed the published values for most other well-characterized AßDPs, including NEP
23
, ECE1
B27 27
, and plasmin
23
, while being comparable to those of IDE
23
B28 28
. Consequently, these values are in good agreement with the side-by-side comparison of Aß degradation in vitro discussed above (Figure 5A).
table
Table 1
Kinetic parameters of Aß40 and Aß42 degradation by BACE2
tgroup align left cols 3
colspec colname c1 colnum 1 colwidth 1*
center c2 2
c3
thead valign top
row rowsep
entry
Aß40
Aß42
tbody
K
M
(μM)
char ±
2.8 ± 0.7
2.3 ± 0.6
V
max
(μM min
-1
)
0.68 ± .083
0.12 ± 0.025
k
cat
(min
-1
)
0.135 ± 0.016
0.025 ± 0.005
k
cat
/
K
M
(M
-1
 min
-1
)
×
4.82 x 107
1.07 x 107
To investigate the relative ability of BACE2 to lower Aß levels under more physiological conditions, we co-transfected CHO cells with APP together with BACE2 or several other AßDPs, then quantified net Aß40 and Aß42 levels in the conditioned medium by ELISA. We emphasize that this approach cannot control for intrinsic differences in transcription or translation efficiency, and, in the case of BACE 2, the Aß-lowering effect can also be mediated to an undetermined degree by BACE2-mediated θ-secretase activity. Nevertheless, the results were in good agreement with the in vitro findings: BACE2 lowered net Aß40 and Aß42 levels to a comparable extent as IDE, with both of the latter being significantly more effective than NEP or plasmin (Figure 5B, C).
BACE2 colocalizes with Aβ intracellularly
Having determined that BACE2 is functionally among the most efficient AßDPs yet discovered, we subsequently investigated the subcellular localization of BACE2, focusing in particular on the extent to which it colocalizes with Aβ in acidic compartments, where BACE2 is expected to be operative. In agreement with other published findings
B29 29
, application of fluorescently tagged Aß to live cells resulted in its accumulation at intracellular sites largely overlapping with lysosomes (Figure F6 6A). To test whether BACE2 is also localized to lysosomes and/or other compartments containing Aß, we analyzed CHO cells expressing BACE2 tagged at its N-terminus with green fluorescent protein (BACE2-GFP). As determined by confocal microscopy, BACE2-GFP was found to be present in lysosomes (Figure 6B) and also to overlap significantly with fluorescently labeled Aß (Figure 6C).
Figure 6BACE2 is localized to intracellular compartments relevant to Aß degradation
BACE2 is localized to intracellular compartments relevant to Aß degradation. A, Exogenous administration of fluorescently labeled Aß40 (green) to CHO cells results in accumulation at intracellular sites overlapping with lysosomes, as labelled by Lysotracker Red (red) and visualized by confocal microscopy. B, BACE2 is expressed in multiple intracellular compartments, including lysosomes. Distribution of GFP-tagged BACE2 (green) in cells labeled with Lysotracker Red (red) shows significant localization within lysosomes (yellow). C, BACE2 colocalizes with exogenously administered Aß. Confocal images showing significant overlap (yellow) between BACE2 (green) and fluorescently labeled Aß (red). For these experiments, cells were imaged within 5 minutes of washing in cold PBS to remove medium containing excess fluorescently labeled Aß. Note that the the majority of BACE2-GFP-expressing cells contianed very low levels of fluorescent Aß (see Figure F7 7), and the particular cell shown exhibited relatively high levels of internalized Aß, allowing us to highlight the overlap with BACE2.
1750-1326-7-46-6
BACE2 degrades Aß at intracellular sites
To directly assess whether BACE2 degrades Aß at intracellular sites, we tested the ability of BACE2-expressing cells to degrade exogenously applied Aß by multiple methods. Cells overexpressing BACE2-GFP and loaded with fluorescently tagged Aß40 showed significantly reduced intracellular Aß 1 h after washing, but this was not the case for cells overexpressing GFP alone (Figure 7A). Consistent with this, levels of intracellular Aß, both fluorescently tagged and unmodified, were found to be consistently lower in cells overexpressing (untagged) BACE2 relative to vector-trasfected controls (Figure 7B,C). Notably, significantly lower levels of intracellular Aß were observed both 5 min and 2 h after washing in multiple paradigms. Collectively, these results strongly suggest that BACE2 is a bona fide AßDP that avidly degrades Aß within acidic compartments.
Figure 7BACE2 degrades Aß at intracellular sites
BACE2 degrades Aß at intracellular sites. A, CHO cells expressing GFP-BACE2 (green, left), but not those expressing GFP alone (green, right), exhibit marked reductions in intracellular Aß (red). For these experiments, cells were loaded for 6 h with 400 nM fluorescently labeled Aß40, washed, then incubated at 37°C for 1 h prior to imaging by conventional fluorescence microscopy. B,C, BACE2 overexpression significantly lowers intracellular Aß. B, Quantification of intracellular pools of fluorescently labeled Aß40 in CHO cells 0 and 2 hours after loading. C, Relative levels of intracellular (unmodified) Aß40 in CHO cells 0 and 2 hours after loading, as quantified by ELISA. Data are mean ± SEM of 3 replicates, normalized to vector-only controls. *P <0.05 by Tukey’s multiple comparisons test.
1750-1326-7-46-7
Conclusions
One of the most fruitful outcomes of the genomic revolution is the emergence of genome-scale collections of full-length, sequence verified cDNAs. Combined with appropriate functional assays, cDNA libraries have catalyzed significant advances in our understanding of AD pathogenesis, including the seminal discovery that ß-secretase activity, the first step in the production of Aß, is mediated by BACE1
21
. Here, we utilized a similar approach to discover new candidate AßDPs, using a functional assay sensitive to both extracellular and intracellular Aß degradation (as well as other potential Aß-lowering mechanisms). Rather unexpectedly, the top hit emerging from a screen of 352 proteases was BACE2, a close homolog of BACE1. Subsequent characterization confirmed that, in addition to BACE2’s established ability to lower Aß production via θ-secretase-mediated processing of APP
11
12
13
14
15
16
, BACE2 also avidly degrades Aß with a catalytic efficiency exceeding almost all well-established AßDPs.The finding that BACE2 is an avid AßDP suggests a novel and unexpected role for this protease in the pathogenesis of AD. Indeed, given its close homology with BACE1, it was initially hypothesized that BACE2 might mediate the production of Aß, via β-secretase cleavage of APP, instead
15
16
. However, most evidence now suggests that BACE2 does not contribute appreciably to Aß production in vivo
3
. For instance, cultured neurons from BACE2 knockout mice did not show reductions in Aß following transfection with APP
B30 30
and conversely, overexpression of BACE2 in APP transgenic mice failed to increase cerebral Aß levels, as would be expected if BACE2 possessed ß-secretase-like activity.In addition to its potent ability to degrade Aß, BACE2 also possesses a second Aß-lowering function for BACE2, one that is quite independent of Aß degradation. Specifically, BACE2 has been shown to cleave APP and the ß-secretase-derived APP-CTF within the Aß sequence, in a manner analogous to α-secretase
11
12
13
14
15
16
. This activity, dubbed θ-secretase
17
, occurs at positions 19 and 20 within the Aß sequence, precisely the same cleavage sites identified in the present study
13
14
. As is true for α-secretase, θ-secretase activity lowers Aß levels by shuttling APP away from the amyloidogenic processing pathway
11
12
13
14
15
16
.As confirmed by previous work
24
, we found that BACE2 also cleaves Aß at the Leu34-Met35 peptide bond, which was in fact the initial and principal site of cleavage. Notably, cleavage at this position can only occur after production of full length Aß by ß- and γ-secretase, because this peptide bond in APP or in APP CTFs is normally embedded within the cell membrane
24
. This fact, together with the finding that Aß34 is produced in cells overexpressing of BACE2 and APP, provides clear evidence that the Aß-degrading activity of BACE2 contributes significantly to the overall Aß-lowering effect of BACE2 overexpression, even in the context of concurrent θ-secretase activity.Given that BACE2 can lower Aß both by decreasing its production and by mediating its degradation, which of these mechanisms are relevant to the pathogenesis or the potential treatment of AD? The answer depends critically on precisely where and to what extent BACE2 is expressed in vivo. Although BACE2 protein is readily detected in brain extracts
15
30
B31 31
B32 32
B33 33
B34 34
B35 35
B36 36
, and its activity has even been shown to be comparable to that of BACE1 in post-mortem brain
31
33
, there is conflicting evidence about which cell types express BACE2. Studies in mice, on the one hand, suggest that the protease is expressed abundantly in glia but only minimally in neurons
30
. To the extent that these findings apply to humans, θ-secretase cleavage of APP by BACE2 would be unlikely to play any significant pathophysiological role in AD, given that APP itself is expressed predominantly in neurons, with only modest expression levels in non-neuronal brain cells
31
. On the other hand, multiple studies in post-mortem human brain tissue have reported detectable BACE2 expression not only in astrocytes, but also in neurons
15
33
, suggesting that the θ-secretase activity of BACE2 may, to some extent, contribute to the overall economy of brain Aß. The pathophysiological relevance of BACE2’s function as an AßDP is similarly difficult to predict and likewise dependent on the extent to which the protease is expressed in neurons. Astrocytes are known mediate the clearance of Aß
B37 37
, but the contribution of intra-astrocytic Aß degradation relative to intraneuronal or extracellular degradation in vivo remains to be established. As was true for other AßDPs first identified in cells
9
, the answer to these questions will require further study in relevant animal models.Notwithstanding uncertainty about its role in AD pathogenesis, a number of considerations suggest that BACE2 represents an especially strong therapeutic candidate, particularly for gene therapy-based approaches. BACE2 can lower Aß catalytically via two independent mechanisms, and its Aß-degrading ability alone exceeds that of most other AßDPs, some of which are being considered for gene therapy clinical trials
B38 38
. Moreover, as an aspartyl protease, BACE2 possesses distinct advantages relative to other AßDPs. First, it is operative with subcellular compartments most relevant to Aß production—i.e., those containing active ß- and γ-secretase, which are both aspartyl proteases—thus allowing it to impact Aß levels prior to secretion. In this connection, there is growing evidence that intracellular Aß may represent an especially pathogenic role in AD
B39 39
, so modulation of this pool may be particularly appropriate therapeutically. Second, because BACE2 is operative exclusively at intracellular sites, its expression could be readily restricted to the site of administration. This is in contrast to many other AßDPs which are secreted and/or active extracellularly
19
B40 40
B41 41
and thus less capable of being confined to specific regions.In conclusion, this study identifies BACE2 as a novel and highly efficient AßDP. This newly identified function of BACE2, together with its established ability to also lower Aß production via θ-secretase activity, suggests that BACE2 may play a significant role in AD pathogenesis. Moreover, even if BACE2 plays no role in the etiology of AD, BACE2 nevertheless represents a particularly attractive candidate for gene therapeutic approaches to the treatment of prevention of this presently incurable disease.
Methods
cDNA screening
A library of 352 full-length, sequence verified, human cDNAs encoding diverse members of all protease classes was purchase from a commercial source (OriGene Technologies, Inc.) in 96-well format (100 ng/well). For negative and positive controls, a subset of blank wells on each plate were supplemented with empty vector or a construct expressing a well-established AßDP, human ECE1b
27
, respectively (100 ng/well). As a source of human Aß and also as a transfection control, each well was cotransfected with a hybrid construct, APP-AP (60 ng/well), comprised of a vector expressing wild-type human APP fused at its N-terminus with alkaline phosphatase (AP)
B42 42
. Additional blank wells were left untreated for cell-free background controls. CHO cells (4.8 x 104/well) suspended in DMEM/Opti-MEM supplemented with 5%FBS were then co-transfected with APP-AP and protease-encoding cDNAs using Fugene 6.0, according to manufacturer’s recommendations (Promega Corp.). Transfected cells were allowed to grow overnight under standard cell culture conditions (5% CO2; 37°C; 95% humidity) then the medium was exchanged. 24 h later, the conditioned media were collected for downstream analysis (see below). All experiments were conducted in compliance with and with approval by the Mayo Clinic Institutional Review Board.
AP activity
Following heat treatment to inactivate endogenous phosphatases (65°C for 15 min) present in the media, conditioned media (30 μL/well) was added to 96-well plates containing AP substrate, 4-nitrophenylphosphate (170 μL/well, 2 mg/mL), dissolved in AP buffer (1 M diethanolamine, 0.5 mM MgCl2, 10 mM L-homoarginine, pH 9.8). Plates were incubated for 30 min and AP activity was determined from absorbance (OD405) using a SpectraMax® M5e multilabel plate reader (Molecular Devices).
Aβ ELISA
Aβ levels were quantified using a sandwich ELISA system based on antibody pairs 33.1.1/13.1.1 for Aβ40 and 2.1.3/4 G8 for Aβ42 as described previously
B43 43
. Conditioned media were supplemented with CompleteTM Protease Inhibitor Cocktail (Roche) just after collection and analyzed immediately. For experiments quantifying intracellular Aß, cells were plated in in 96-well plates (2 x 104 cells per well) and transfected with BACE2-encoding cDNA or empty vector, washed, then incubated with 400 nM synthetic Aß for 6 h. After washing with PBS, intracellular Aß was extracted with 5 M guanidinium isothiocyanate and quantified using a commercially available ELISA (Wako Chemicals USA, Inc.) after 10-fold dilution in the manufacturer-provided dilution buffer.
Mass spectrometry
The cleavage sites within Aβ40 and Aβ42 hydrolyzed by BACE2 and BACE1 were determined essentially as described
B44 44
with minor modifications. Briefly, Aβ peptides or biotinylated Aβ peptides were incubated for various lengths of time with recombinant BACE2 enzyme in Assay Buffer (25 mM acetate buffer, pH 4.0, supplemented with 0.1% BSA). The reaction was stopped by addition of protease inhibitor cocktail and pH adjustment. Aβ fragments were immediately precipitated by magnetic beads coated with streptavidin (for biotinylated Aβ) or magnetic beads coated with Ab9 antibody
B45 45
(for unmodified Aβ). Beads were washed with 10 mM NH4CO3, pH 8.0, and peptide fragments were eluted using 0.5% trifluoroacetic acid in 75% acetonitrile in water, followed by the addition of an equal volume of a saturated sinapic acid solution dissolved in 0.5% trifluoroacetic acid in 50% acetonitrile and water. Digested products were spotted onto a gold chip, dried, and analyzed using a Ciphergen ProteinChip SELDI time-of-flight system (Bio-Rad). Mass spectra were acquired automatically in a linear positive mode at 1350 shots per spectrum. Peptides containing a183-Da increase in MW were identified as being modified by AEBSF, as previously reported
46
. Same procedure was applied to detect the endogenous Aβ fragments produced by CHO cells transfected with APP and BACE2 (using Ab9 as a capture antibody).
In vitro analyses of Aβ degradation by BACE2
The kinetics of Aβ40 and Aβ42 degradation by BACE2 were determined using freshly prepared, monomeric Aβ peptides separated from aggregated species by size-exclusion chromatography and characterized as described
25
26
. Aβ peptides were diluted in neutral Dilution Buffer (20 mM Tris, pH 8.0 supplemented with 0.1% BSA) and reactions were initiated by transfer into 20 times more volume of Assay Buffer supplemented with purified recombinant BACE2 (R&D Systems, nominal concentration 1 or 5 nM) or, as a control for non-specific loss of Aβ, the latter buffer lacking BACE2. Where required, reactions were terminated by supplementation with protease inhibitor cocktail and adjustment to neutral pH. For ELISA-based experiments, Aβ42 and Aβ40 were quantified using the sandwich ELISAs described above. For determination of kinetic parameters, ELISAs were used to quantify the initial velocities of degradation of a range of different concentrations of Aß40 (0.2 to 16 μM) or Aß42 (0.6 to 16 μM) by a fixed amount of recombinant BACE2 (5 nM) in Assay Buffer, and K
M and v
max values were determined in triplicate by fitting a hyperbolic curve to these data in Prism 5.0 (GraphPad Software, Inc.). For determination of the pH dependence of Aβ degradation, experiments were carried same as described above, using Assay Buffer at different pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5). The reactions were stopped at 10 min and the remaining 200nM of Aβ was determined using a well-characterized fluorescence polarization-based activity Aß degradation assay, as described
23
. For comparison of the rate of degradation of Aβ by different proteases, we incubated 200 nM of Aβ fluorescent substrate (FAβB) with 5nM of different protease in their corresponding buffers: BACE1 and BACE2 using Assay Buffer and IDE, NEP, and plasmin in PBS, pH 7.4 supplemented with 0.1% BSA. The reactions were stopped by addition of protease inhibitor cocktail, 500nM streptavidin, and adjustment to neutral pH. The degree of Aβ hydrolysis was immediately determined using a polarization-based Aß degradation assay
23
. Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described
23
. All reactions were performed at 37°C.
Fluorescence microscopy
CHO cells (106 cells/cm2) were plated onto 8-well poly-D-lysine-coated, glass-bottom chambers (MatTek Corp.) in culture medium (DMEM/Opti-MEM supplemented with 5%FBS). For BACE2 transfections, cell were transfected with a construct encoding BACE2 tagged at its C-terminus with GFP (OriGene Technologies, Inc. Cat. No. RG04860) using Fugene 6.0 transfection reagent according to manufacturer’s recommendations (Promega Corp.). For Aβ colocalization experiments, cells were washed twice in fresh culture medium, then incubated in the latter medium supplemented with either Aß40 (500 nM) labeled at the N-terminus with HiLight FluorTM 488 or HiLight FluorTM 555 (AnaSpec, Inc.). For lysosomal staining, cells were incubated with Lysotracker Red according to manufacturer’s recommendations (Invitrogen Corp.), then washed 2 times with fresh culture medium prior to imaging. For confocal microscopy, cells were washed with fresh medium then imaged immediately using the 488-nm and 543-nm laser lines on a Zeiss LCM 510 META confocal microscope (Carl Zeiss, Inc.). Images were processed and analyzed using MetaMorph software according to manufacturer’s recommendations (Molecular Devices, Inc.). For conventional fluorescence microscopy of intracellular Aß, cells were washed with fresh medium, then incubated at 37°C for 1 h prior to imaging using a Nikon Labophot 2 fluorescent microscope (Nikon Inc.).
Abbreviations
Aß: Amyloid ß-protein; AßDP: Aß-degrading protease; AD: Alzheimer disease; AP: Alkaline phosphatase; APP: Amyloid precursor protein; BACE1: ß-site APP-cleaving enzyme-1; BACE2: ß-site APP-cleaving enzyme-2; ECE1: Endothelin-converting enzyme-1; IDE: Insulin-degrading enzyme; NEP: Neprilysin.
Competing interests
The authors declare they have no competing interests.
Authors' contributions
SA-H contributed to the design of experiments, executed the screen and all follow up experiments, analyzed data, and drafted the manuscript. TS assisted with the execution of the primary screen. MM and DK assisted with the maintenance of cell cultures. ML conceived of the experimental approach, designed experiments, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
We thank Dr. Terrone Newberry and William Tay for providing monomeric and fibrillar Aß peptides and Drs. Todd Golde and Kevin Felsenstein for contributing the APP-AP construct. Supported by a grant from the Coins For Alzheimer’s Trust Fund to ML.
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Abstract
Background
Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer’s disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays.
Results
The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds—Phe19-Phe20, Phe20-Ala21, and Leu34-Met35—with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE.
Conclusions
This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.
http:purl.orgdcelements1.1creator
Abdul-Hay, Samer O
Sahara, Tomoko
McBride, Melinda
Kang, Dongcheul
Leissring, Malcolm A
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BioMed Central Ltd
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Samer O Abdul-Hay et al.; licensee BioMed Central Ltd.
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ui 1479-7364-6-9
ji 1479-7364
fm
dochead Primary research
bibl
title
p CXCL5 polymorphisms are associated with variable blood pressure in cardiovascular disease-free adults
aug
au id A1 ca yes ce snm Beitelsheesmi Lfnm Amberinsr iid I1 email abeitels@medicine.umaryland.edu
A2 AquilanteLChristinaI2 christina.aquilante@ucdenver.edu
A3 AllayeeHoomanI3 hallayee@usc.edu
A4 LangaeeYTaimourI4 langaee@cop.ufl.edu
A5 WelderJGregorygw21stunna@gmail.com
A6 SchofieldSRichardI5 richard.schofield@medicine.ufl.edu
A7 ZinehIssamIssam.Zineh@fda.hhs.gov
insg
ins Division of Endocrinology, Diabetes and Nutrition, University of Maryland School of Medicine, 660 W. Redwood St, HH469, Baltimore, MD, 21201, USA
Department of Pharmaceutical Sciences, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, 80045, USA
Department of Preventive Medicine and Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA
Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, University of Florida College of Pharmacy, Gainesville, FL, 32610, USA
Division of Cardiovascular Medicine and Department of Veterans Affairs Medical Center, University of Florida College of Medicine, Gainesville, FL, 32603, USA
source Human Genomics
issn 1479-7364
pubdate 2012
volume 6
issue 1
fpage 9
url http://www.humgenomics.com/content/6/1/9
xrefbib pubid idtype doi 10.1186/1479-7364-6-9
history rec date day 18month 5year 2012acc 1852012pub 282012
cpyrt 2012collab Beitelshees et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd CXCL5
ENA-78
Blood pressure
Hypertension
Leukocytes
abs
sec
st
Abstract
Objective
Leukocyte count has been associated with blood pressure, hypertension, and hypertensive complications. We hypothesized that polymorphisms in the it CXCL5 gene, which encodes the neutrophilic chemokine ENA-78, are associated with blood pressure in cardiovascular disease (CVD)-free adults and that these polymorphisms are functional.
Methods and results
A total of 192 community-dwelling participants without CVD or risk equivalents were enrolled. Two CXCL5 polymorphisms (−156 G > C (rs352046) and 398 G > A (rs425535)) were tested for associations with blood pressure. Allele-specific mRNA expression in leukocytes was also measured to determine whether heterozygosity was associated with allelic expression imbalance. In −156 C variant carriers, systolic blood pressure (SBP) was 7 mmHg higher than in −156 G/G wild-type homozygotes (131 ± 17 vs. 124 ± 14 mmHg; P = 0.008). Similarly, diastolic blood pressure (DBP) was 4 mmHg higher in −156 C variant carriers (78 ± 11 vs. 74 ± 11 mmHg; P = 0.013). In multivariate analysis of SBP, age, sex, body mass index, and the −156 G > C polymorphism were identified as significant variables. Age, sex, and the −156 G > C SNP were further associated with DBP, along with white blood cells. Allelic expression imbalance and significantly higher circulating ENA-78 concentrations were noted for variant carriers.
Conclusion
CXCL5 gene polymorphisms are functional and associated with variable blood pressure in CVD-free individuals. The role of CXCL5 as a hypertension- and CVD-susceptibility gene should be further explored.
bdy
Introduction
The relationship between inflammation and elevated blood pressure is increasingly being evaluated
abbrgrp
abbr bid B1 1
B2 2
. It has been shown that elevated concentrations of prototypical pro-inflammatory markers such as interleukin-6, C-reactive protein (CRP), and tumor necrosis factor-alpha are associated with increased blood pressure, incidence of hypertension, and the likelihood for hypertensive complications
B3 3
B4 4
B5 5
B6 6
B7 7
B8 8
B9 9
B10 10
B11 11
B12 12
B13 13
B14 14
. It has been further suggested that this inflammatory-hypertensive relationship results from increased number or activity of common cellular mediators such as white blood cells (WBC)
B15 15
B16 16
. For example, studies have demonstrated elevated WBC count to be associated with increased incident hypertension as well as increased blood pressure within the normal to pre-hypertensive range
B17 17
B18 18
B19 19
B20 20
B21 21
B22 22
.
Although the exact mechanistic relationship between leukocytosis and elevated blood pressure is unknown, it is plausible that low-grade inflammation may be a contributing factor. In this regard, WBC count may be a surrogate marker for increased activation of inflammatory pathways that cause leukocyte recruitment and activation. As such, increased activity of leukocytic chemokines could be related to increased blood pressure.
Epithelial neutrophil activator-78 (ENA-78), a key leukocytic chemokine that is both a neutrophil attractor and activator, has been implicated in many diseases with an inflammatory component (e.g., obesity, diabetes, subclinical atherosclerosis, acute coronary syndromes)
B23 23
B24 24
B25 25
B26 26
B27 27
B28 28
B29 29
B30 30
B31 31
B32 32
. We have previously reported that two single nucleotide polymorphisms (SNPs), -156 G > C (rs352046) and 398 G > A (rs425535), in the gene encoding ENA-78 (CXCL5) occur in sites important for transcription and exon splicing
B33 33
. In our previous work, a relationship existed between these SNPs and both plasma concentrations and leukocyte production of the ENA-78 chemokine protein
33
. We then went on to show an association between the CXCL5 -156 G > C polymorphism and worse outcomes in patients with acute coronary syndromes
27
. In the present work, to the extent that ENA-78 is important in neutrophil recruitment and degranulation, we hypothesized that one or both of these polymorphisms (−156 G > C and 398 G > A) could be associated with differences in blood pressure in individuals without established cardiovascular disease (CVD). Specifically, we hypothesized that relatively young individuals without known CVD who were carriers of CXCL5 variant alleles would exhibit higher systolic blood pressure (SBP), diastolic blood pressure (DBP), or pulse pressure (PP) than wild-type homozygotes. Furthermore, to assess whether there was a functional role for these polymorphisms, we measured allele-specific mRNA expression of CXCL5 in leukocytes obtained from CVD-free individuals who were heterozygous for the SNPs at both loci.
Materials and methods
Study population
The study population has been previously described
33
. Briefly, participants were recruited from two sites in the USA and had to be at least 18 years of age without known CVD or CVD-risk equivalents (e.g., diabetes, peripheral vascular disease, 10-year Framingham Risk ≥20%) as defined by National Cholesterol Education Program criteria
B34 34
. Other exclusions were pregnancy, malignancy, substance abuse, and routine use of medications known to affect WBC counts such as systemic steroids and other anti-inflammatory agents. Individuals were excluded from analysis if they were taking anti-hypertensive medications for either cardiovascular or non-cardiovascular indications (e.g., migraine). For blood pressure measurement, subjects were seated for at least 5 min in a quiet, temperature-controlled General Clinical Research Center (GCRC) outpatient clinic room, and two blood pressure measurements were taken at least 5 min apart. The average of the duplicate blood pressure measurements was used for this investigation. Blood samples were obtained from participants enrolled in University of Florida- and Colorado Multiple Institutional Review Board (IRB)-approved studies. All subjects provided written informed consent to specimen and data use in genetic association and related studies.
Genotype and inflammatory biomarker determination
Genomic DNA was isolated from whole blood or buccal cells using previously described methods
B35 35
. CXCL5 genotypes were determined by polymerase chain reaction (PCR) and pyrosequencing (Qiagen, Valencia, CA, USA) as we have previously described
B36 36
. Circulating high-sensitivity CRP (as a non-specific marker of inflammation) was measured by the Shands Hospital Laboratory at the University of Florida and University of Colorado GCRC. ENA-78 concentrations were measured by cytometric fluorescence detection as previously described (Luminex™100 IS system; Luminex Corp., Austin, TX, USA; Fluorokine® MAP Multiplex Human Cytokine Panel A; R&D Systems, Minneapolis, MN, USA)
B37 37
. Samples were stored at −80°C until CRP and ENA-78 detection was performed.
Allele-specific mRNA quantification
To determine whether variant carrier status results in functional changes at the transcriptional level, we quantified allele-specific mRNA transcripts from leukocytes using pyrosequencing-based methodology
B38 38
B39 39
. Specifically, the presence or absence of allelic expression imbalance was determined using leukocytes obtained from 18 individuals who were heterozygotic for both the −156 G > C and 398 G > A polymorphisms. The 398 G > A SNP was chosen as the genetic biomarker in these experiments because it is located in the coding region of CXCL5, while −156 G > C is a promoter polymorphism and as such cannot be quantified at the mRNA level. Because of the near complete linkage of the studied SNPs, we chose individuals who were heterozygotes at both loci so that 398 G > A genotype might serve as a functional surrogate for the upstream promoter locus.
Leukocyte mRNA was prepared from approximately 6 × 10sup 6 cells from each individual using the RNeasy mini kit (Qiagen, Valencia, CA, USA). Cells were rinsed, lysed, and homogenized in buffered solutions and subsequently passed through the RNeasy mini column (Qiagen, Valencia, CA, USA). Following a series of washes at room temperature and 15-min incubation with DNase, concentrations were determined by spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized using approximately 450 ng of cellular RNA from each individual using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) per protocol. Conditions for reverse transcription were 25°C for 10 min followed by 37°C for 2 h. cDNA quality was assessed by comparing cDNA and DNA PCR products generated using intron-spanning primers by gel electrophoresis. For allele-specific transcript quantification, subject DNA and cDNA underwent PCR simultaneously using previously described conditions
36
. PCR products obtained for genotype determination (DNA) and transcript quantification (mRNA) were assayed in parallel pyrosequencing reactions to minimize cycle variability. Pyrosequencing analyses were performed in duplicate on three separate PCR amplification products, and the results were pooled for analysis. Peak heights were determined by the pyrosequencing allele quantification algorithm. In genomic DNA, the ratio of 398A:G alleles for DNA in heterozygotes is expected to be approximately 1, whereas significant deviations from this ratio in mRNA would suggest allele expression imbalance associated with the variant allele.
Statistical analyses
Genotype frequencies were determined by allele counting, and departures from Hardy-Weinberg equilibrium were assessed by chi-square analyses. Differences in blood pressure by genotype groups (0, homozygous for common allele; 1, heterozygous or homozygous for variant allele) were compared using one-way ANOVA. Based on the preexisting sample size and prevalence of variant alleles, we had 80% power with a two-sided α of 0.05 to detect a 6-mmHg difference in SBP, 4-mmHg difference in DBP, and 4-mmHg difference in PP between genotype groups. Multiple regression analysis was performed if blood pressure differences were seen across genotype groups. Covariates for multiple regression were chosen through univariate analyses of age, sex, smoking status (0, non-smoker; 1, current smoker), body mass index (BMI), CRP concentration, ENA-78 concentration, and WBC count. Any variable with a P ≤ 0.1 on univariate analysis was entered into the multivariable model. Because of small numbers of individuals within racial groups, analyses could not be performed within racial strata. However, race (0, white; 1, non-white) was included in all multivariable analyses, and a race-by-genotype interaction term was considered in the regression models to avoid spurious associations secondary to racial differences in allele frequency. Multiple regression using step-type selection methods was performed to determine the joint effects of CXCL5 genotypes and clinical variables on SBP, DBP, or PP. All statistical analyses were performed using SPSS (version 11.5, SPSS Inc., Chicago, IL, USA) or SAS (version 9.1, SAS Institute Inc., Cary, NC, USA). A P value < 0.05 was considered statistically significant.
Results
Baseline demographic characteristics are shown in Table 
tblr tid T1 1. Participants were on average 39 ± 12 years old with blood pressures of 126/75 ± 15/11 mm Hg. -156 G > C and 398 G > A genotypes were determined for 189 and 188 of the 192 individuals, respectively. The overall −156 C and 398A minor allele frequencies were both 15%. Variant allele frequencies differed by race whereby the −156 C allele frequency was 14%, 45%, and 11%, and 398A allele frequency was 13%, 46%, and 9% in Caucasians, blacks, and non-black Hispanics, respectively. Genotype distributions satisfied criteria for Hardy-Weinberg equilibrium (data not shown). The two SNPs were in a high degree of linkage disequilibrium with r
2 for Caucasian, black, and Hispanic individuals of 0.82, 1.0, and 0.51, respectively, in our study population.
table
Table 1
caption
b Baseline characteristics
tgroup align left cols 2
colspec colname c1 colnum 1 colwidth 1*
c2
thead valign top
row rowsep
entry
Characteristic
N
 = 192
tfoot
aTotal, HDL, and triglycerides available in 94% of subjects; LDL available in 92% of subjects. bCRP and ENA-78 available for 88% and 91% of subjects, respectively.
tbody
Age (mean ± SD, years)
39 ± 12
Women (number (%))
124 (65)
Race/ethnicity (number (%))
 White
148 (77)
 Black
12 (6)
 Hispanic
19 (10)
 Other
13 (7)
Family heart disease history (number (%))
29 (15.1)
Smoking (number (%))
35 (18)
Body mass index (mean ± SD, kg/m2)
29.6 ± 7
Blood pressure (mean ± SD, mmHg)
 Systolic
126 ± 15
 Diastolic
75 ± 11
Pulse pressure (mean ± SD, mmHg)
51 ± 10
Cholesterol (mean ± SD, mg/dLa)
 Total
201 ± 43
 LDL
118 ± 36
 HDL
55 ± 17
Triglycerides
139 ± 107
White blood cell count, (mean ± SD, ×109 cells/L)
6.3 ± 2.0
C-reactive protein (median (range), mg/Lb)
1.78 (0.1–16.9)
ENA-78 (median (range), pg/mLb)
362 (32.2–3970)
Genotype association with blood pressure
In −156 C variant carriers, SBP was 7-mmHg higher than in −156 G/G wild-type homozygotes (131 ± 17 vs. 124 ± 14 mmHg; P = 0.008). Similarly, DBP was 4-mmHg higher in −156 C variant carriers (78 ± 11 vs. 74 ± 11 mmHg; P = 0.013). PP did not differ between −156 C variant carriers and wild-type homozygotes (53 ± 11 vs. 51 ± 10; P = 0.22). Because of the high degree of linkage disequilibrium between the 398 G > A and −156 G > C SNPs, blood pressure differences were similar when compared by 398 G > A genotypes. For example, SBP was 130 ± 16 and 125 ± 14 mmHg in 398A variant carriers and 398 G/G homozygotes, respectively (P = 0.033); DBP was 78 ± 11 and 74 ± 11 mmHg, respectively (P = 0.038); and PP was not different between groups (53 ± 11 vs. 51 ± 10 mmHg in 398A carriers and 398 G/G homozygotes, respectively; P = 0.362).
Age (P ≤ 0.001), sex (P ≤ 0.008), and BMI (P ≤ 0.002) were common univariate predictors of SBP, DBP, and PP. Furthermore, WBC count (P = 0.10 for SBP; P = 0.076 for DBP) and both CXCL5 polymorphisms (range P = 0.008 to 0.038) were additional predictors of SBP and DBP, while smoking status was associated with SBP alone (P = 0.038). In terms of circulating CRP and ENA-78 levels, both biomarkers were significant for SBP (P = 0.005 for CRP and P = 0.033 for ENA-78) and PP (P = 0.001 for CRP and P = 0.007 for ENA-78) in univariate analyses. Consistent with our previous report, CXCL5 genotype was associated with ENA-78 protein concentrations in the plasma whereby variant carriers at either SNP locus had higher protein concentrations than wild-type homozygotes (P = 0.003; Figure
figr fid F1 1).
fig Figure 1Plasma ENA-78 by CXCL5 -156 G  C genotype/p/captiontext
pbPlasma ENA-78 by /bbitCXCL5 /it/bb-156 G  C genotype. bitPit = 0.003; data were similar for the exon 2 SNP, data not shown (itPit = 0.001).p
textgraphic file="1479-7364-6-9-1"fig
pIn multivariate analysis of SBP, age, sex, BMI, and the itCXCL5it -156 G > C promoter polymorphism were identified as significant variables (Table 
tblr tid="T2"2tblr). The overall model that included these variables explained 32.5% of the variability in SBP (itPit < 0.001). Consideration of the 398 G > A polymorphism rather than the −156 G > C promoter SNP resulted in a model in which only age, sex, and BMI were significantly associated with SBP (itRit
sup2sup = 0.301; itPit < 0.001).p
table id="T2"
title
pTable 2p
title
caption
p
bMultivariate predictors of systolic blood pressure in cardiovascular disease-free individualsb
p
caption
tgroup align="left" cols="4"
colspec align="left" colname="c1" colnum="1" colwidth="1*"
colspec align="center" colname="c2" colnum="2" colwidth="1*"
colspec align="center" colname="c3" colnum="3" colwidth="1*"
colspec align="center" colname="c4" colnum="4" colwidth="1*"
thead valign="top"
row rowsep="1"
entry colname="c1"
p
bVariableb
p
entry
entry colname="c2"
p
b
itβit
b
p
entry
entry colname="c3"
p
bStandard errorb
p
entry
entry colname="c4"
p
b
itPit
bbvalueb
p
entry
row
thead
tfoot
p
itRit
sup2sup = 0.325; itPit < 0.0001.p
tfoot
tbody valign="top"
row
entry colname="c1"
pConstantp
entry
entry colname="c02"
p100p
entry
entry colname="c3"
p4.86p
entry
entry colname="c4"
p<0.0001p
entry
row
row
entry colname="c1"
pAgep
entry
entry colname="c2"
p0.313p
entry
entry colname="c3"
p0.094p
entry
entry colname="c4"
p0.001p
entry
row
row
entry colname="c1"
pSexp
entry
entry colname="c2"
p−9.84p
entry
entry colname="c3"
p2.12p
entry
entry colname="c4"
p<0.0001p
entry
row
row
entry colname="c1"
pBMIp
entry
entry colname="c2"
p0.637p
entry
entry colname="c3"
p0.160p
entry
entry colname="c4"
p<0.0001p
entry
row
row rowsep="1"
entry colname="c1"
p−156 C carrierp
entry
entry colname="c2"
p4.93p
entry
entry colname="c3"
p2.30p
entry
entry colname="c4"
p0.034p
entry
row
tbody
tgroup
table
pAge, sex, and the −156 G > C SNP were further associated with DBP, along with WBC (Table 
tblr tid="T3"3tblr). Consideration of this promoter SNP (model itRit
sup2sup = 0.168; itPit < 0.0001) was slightly more informative than consideration of the 398 G > A SNP (itPit = 0.067) in which case age (itPit < 0.0001), sex (itPit = 0.001), and WBC (itPit = 0.02) still remained significant (model itRit
sup2sup = 0.145; itPit < 0.0001). In multivariable models of PP, only sex (itPit < 0.004) and BMI (itPit < 0.0001) were significant (model itRit
sup2sup = 0.247; itPit < 0.0001).p
table id="T3"
title
pTable 3p
title
caption
p
bMultivariate predictors of diastolic blood pressure in cardiovascular disease-free individualsb
p
caption
tgroup align="left" cols="4"
colspec align="left" colname="c1" colnum="1" colwidth="1*"
colspec align="center" colname="c2" colnum="2" colwidth="1*"
colspec align="center" colname="c3" colnum="3" colwidth="1*"
colspec align="center" colname="c4" colnum="4" colwidth="1*"
thead valign="top"
row rowsep="1"
entry colname="c1"
p
bVariableb
p
entry
entry colname="c2"
p
b
itβit
b
p
entry
entry colname="c3"
p
bStandard errorb
p
entry
entry colname="c4"
p
b
itPit
bbvalueb
p
entry
row
thead
tfoot
p
itRit
sup2sup = 0.168; itPit < 0.0001.p
tfoot
tbody valign="top"
row
entry colname="c1"
pConstantp
entry
entry colname="c2"
p63.13p
entry
entry colname="c3"
p3.42p
entry
entry colname="c4"
p<0.0001p
entry
row
row
entry colname="c1"
pAgep
entry
entry colname="c2"
p0.247p
entry
entry colname="c3"
p0.063p
entry
entry colname="c4"
p<0.0001p
entry
row
row
entry colname="c1"
pSexp
entry
entry colname="c2"
p−5.801p
entry
entry colname="c3"
p1.549p
entry
entry colname="c4"
p<0.0001p
entry
row
row
entry colname="c1"
p−156 C carrierp
entry
entry colname="c2"
p3.735p
entry
entry colname="c3"
p1.630p
entry
entry colname="c4"
p0.023p
entry
row
row rowsep="1"
entry colname="c1"
pWBCp
entry
entry colname="c2"
p0.768p
entry
entry colname="c3"
p0.374p
entry
entry colname="c4"
p0.041p
entry
row
tbody
tgroup
table
sec
sec
st
pAllelic expression imbalancep
st
pAllele-specific mRNA quantification was performed to determine whether there is a functional basis for the differences seen in blood pressure based on itCXCL5it genotypes (see ‘Materials and methods’ section for rationale of 398 G > A as marker SNP). Importantly, there was consistently higher expression of itCXCL5it mRNA from the 398A allele compared to the 398 G allele in heterozygous individuals (Figure
figr fid="F2"2figrA). For example, individual heterozygotes displayed anywhere from 2.2-fold to 3.4-fold higher expression of 398A variant transcripts compared to the 398 G allele, with a mean ratio of 2.9 (Figure
figr fid="F2"2figrB; itPit = 7.4E-15).p
fig id="F2"titlepFigure 2ptitlecaptionpAllele-specific CXCL5 mRNA expression in leukocytespcaptiontext
pbAllele-specific bbitCXCL5 itbbmRNA expression in leukocytes.b (bAb) Allelic mRNA and DNA ratios were measured in 18 cardiovascular disease-free individuals heterozygous for the 398 G  A SNP. The AG ratios in DNA were close to 1 suggesting equal abundance of both alleles, whereas there was consistently higher expression of mRNA from the 398A allele compared to the 398 G allele. (bBb) Pooled 398AG ratios from 18 heterozygous individuals. The sample displayed 2.9-fold higher expression of 398A variant transcripts compared to the 398 G allele (itPit = 7.4E-15). Data are presented as mean ± SDp
textgraphic file="1479-7364-6-9-2"fig
sec
sec
sec
st
pDiscussionp
st
pAccumulating evidence points to a relationship between inflammation and blood pressure. Data suggest that WBC counts are associated with incident hypertension and correlated with blood pressure concentrations. We hypothesized that WBC count is a surrogate for leukocytic chemokine activity and that the itCXCL5it gene, which encodes the neutrophil attractor ENA-78, may be an important determinant of blood pressure. We demonstrated a significant, independent relationship between itCXCL5it polymorphisms and SBP and DBP in the overall population of CVD-free individuals. Variant carriers of the −156 G > C promoter SNP had 7-mmHg and 4-mmHg higher SBP and DBP, respectively, than those with the wild-type −156 GG genotype. Because of the epidemiologically significant difference in CVD risk conferred by blood pressure differences of this magnitude, and since variant carriers represent approximately 30% of the population studied, itCXCL5it polymorphisms should be considered as a potential novel biomarker of pre-hypertension, hypertension, and CVD risk requiring future study. However, it is important to emphasize that genetic associations are preliminary and will require confirmation in additional populations.p
pOf particular interest, WBC count (along with traditional variables such as age, sex, smoking status, and BMI) was significantly associated with SBP and DBP in univariate analysis among CVD-free individuals. This finding supports the report by Orakzai et al. that demonstrated a relationship between WBC counts and SBP among nearly 3,500 white individuals without CVD and with SBP < 140 mmHg on entry
abbrgrp
abbr bid="B20"20abbr
abbrgrp. It also supports data from other clinical cohorts showing an association between WBC count, major WBC components (e.g., neutrophils), and blood pressure
abbrgrp
abbr bid="B21"21abbr
abbr bid="B22"22abbr
abbr bid="B40"40abbr
abbr bid="B41"41abbr
abbrgrp. However, in our analysis WBC count was no longer a significant predictor of SBP when itCXCL5it genotype was included in multivariable analysis, suggesting genotype may capture the contribution of inflammation to SBP more effectively than WBC count. WBC did, however, remain a significant predictor of DBP in multivariate analysis, along with age, sex, and itCXCL5it -156 G > C genotype.p
pTo determine whether there is any functional basis for an observed association between itCXCL5it variant alleles and blood pressure, we performed allele expression imbalance experiments in a subset of participants. The exonic 398 G > A allele was chosen as the genetic marker given its location in the coding region of the mRNA. However, the 398 GA heterozygous individuals (itNit = 18) were also heterozygous for the promoter polymorphism, which minimizes confounding of an association by differing genotypes at the upstream locus. It was noted that variant carriers displayed nearly threefold higher expression of variant itCXCL5it mRNA transcripts from the 398A allele. This novel finding is consistent with our previous observation that variant carriers exhibited higher plasma and leukocyte-produced ENA-78 than wild-type homozygotes and that the promoter and exonic SNPs occur in transcription factor binding and splicing enhancer sites, respectively
abbrgrp
abbr bid="B33"33abbr
abbrgrp. Given that the −156 G > C and 398 G > A SNPs are in near perfect linkage disequilibrium, it is unclear which polymorphism is the causal variant and functionally contributes to the blood pressure phenotype. However, the −156 G > C promoter SNP was more significantly correlated with blood pressure in our study. Further functional studies of these SNPs are warranted.p
pIn addition to genotype and traditional covariates, we included plasma CRP and ENA-78 protein concentrations in our analyses. While CRP and ENA-78 were significantly associated with SBP (and PP) in univariate analyses, they fell out of the models when itCXCL5it genotype was included. This suggests that in our analyses, genotype is more significantly associated with the blood pressure phenotype than systemically circulating concentrations of the non-specific inflammatory mediator CRP and the itCXCL5it protein product ENA-78. While this observation may appear somewhat contradictory, it can be postulated that itCXCL5it gene polymorphisms may be better indicators of chemokine activity at the target organ (e.g., endothelium) level than a measurement in the circulation. Because of trans-acting influences on systemic biomarker expression, polymorphisms in itCXCL5it may be more robustly associated with blood pressure. In fact, we have shown a similar finding in a different population for the endothelial nitric oxide synthase gene where itNOS3it gene polymorphisms, but not measures of circulating NO activity, were associated with arterial stiffness in children with type 1 diabetes
abbrgrp
abbr bid="B42"42abbr
abbr bid="B43"43abbr
abbrgrp. Further support for this observation can be found in a case–control study of the role of ENA-78 in patients with ischemic stroke. Zaremba et al. demonstrated that serum ENA-78 protein concentrations were not different between stroke patients and controls; contrarily, it was demonstrated that ENA-78 concentrations were significantly higher (twofold) in the cerebrospinal fluid of stroke patients compared with controls
abbrgrp
abbr bid="B44"44abbr
abbrgrp. Taken in sum, it is possible that genotype more effectively captures the likelihood for local preponderance of chemokine activity than plasma protein level.p
pIn general, there is biological plausibility for the role of itCXCL5it in CVD. For example, the protein product of itCXCL5it, ENA-78, belongs to the same class of chemokines as IL-8, IP-10, and I-TAC, which have been previously implicated in atherosclerotic inflammation
abbrgrp
abbr bid="B23"23abbr
abbr bid="B45"45abbr
abbrgrp. ENA-78 has been shown to be chemotactic for neutrophils and stimulate neutrophilic degranulation causing release of myeloperoxidase and generating reactive oxygen species
abbrgrp
abbr bid="B24"24abbr
abbr bid="B25"25abbr
abbrgrp. In addition, ENA-78 is involved in platelet-dependent activation of monocytes, displays angiogenic properties, and has been implicated in diseases such as obesity, diabetes, subclinical atherosclerosis, acute coronary syndromes, ischemic stroke, abdominal aortic aneurysm, and thrombosis
abbrgrp
abbr bid="B27"27abbr
abbr bid="B28"28abbr
abbr bid="B32"32abbr
abbr bid="B44"44abbr
abbr bid="B46"46abbr
abbr bid="B47"47abbr
abbr bid="B48"48abbr
abbr bid="B49"49abbr
abbr bid="B50"50abbr
abbr bid="B51"51abbr
abbrgrp. Hypertension is a risk factor for adverse events such as atherosclerosis, stroke, and abdominal aortic aneurysm, and ENA-78 is overexpressed in these situations. We have shown itCXCL5it polymorphisms to be associated with ENA-78 concentrations, blood pressure, and prognosis following acute coronary syndromes
abbrgrp
abbr bid="B27"27abbr
abbr bid="B33"33abbr
abbrgrp. Thus, the role of itCXCL5it in CVD should be further explored. As final hypothesis-generating evidence of a link between the itCXCL5it pathway and blood pressure, statins have been hypothesized to have mild antihypertensive effects, and we have shown that atorvastatin reduces ENA-78 production from human endothelial cells in a dose-dependent fashion
abbrgrp
abbr bid="B52"52abbr
abbr bid="B53"53abbr
abbrgrp. Our findings, along with existing data, support the need for future investigation of itCXCL5it as a hypertension- and CVD-susceptibility gene.p
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pCompeting interestsp
st
pThe authors declare that they have no competing interests.p
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pAuthors’ contributionsp
st
pALB performed statistical analyses and drafted the manuscript. CLA enrolled the study subjects and drafted the manuscript. HA assisted in the molecular genetic studies and provided critical revision of the manuscript. TYL assisted in the molecular genetic studies. GJW assisted in the molecular genetic studies. RSS assisted with the clinical study. IZ conceived the manuscript, enrolled the study subjects, and drafted the manuscript. All authors read and approved the final manuscript.p
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pAcknowledgmentsp
st
pWe thank Dr. Julie A. Johnson for her thoughtful comments regarding the manuscript. We thank Lauren Burt and Lynda Stauffer for their laboratory assistance. This work was supported by American Heart Association FloridaPuerto Rico Affiliate Scientist Development Grant 0435278B, American College of Clinical Pharmacy Kos Dyslipidemia Research and Pharmacotherapy New Investigator Awards, American Association of Colleges of Pharmacy New Investigator Program Award, the University of Colorado Denver General Clinical Research Center (RR00051), and NIH C06 Grant RR17568. ALB is supported by K23 HL091120.p
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