Hepatocyte growth factor and carotid intima-media thickness in relation to circulating CD34-positive cell levels
Shimizu et al. Environmental Health and Preventive Medicine
Hepatocyte growth factor and carotid intima-media thickness in relation to circulating CD34-positive cell levels
Yuji Shimizu 0 1
Shimpei Sato 1
Jun Koyamatsu 2
Hirotomo Yamanashi 2
Mako Nagayoshi 1
Shin-Ya Kawashiri 1
Keita Inoue 1
Shoichi Fukui 1
Hideaki Kondo 1
Seiko Nakamichi 4
Yasuhiro Nagata 3
Takahiro Maeda 1 2
0 Department of Cardiovascular Disease Prevention, Osaka Center for Cancer and Cardiovascular Disease Prevention , Osaka , Japan
1 Department of Community Medicine, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki-shi, Sakamoto 1-12-4, Nagasaki 852-8523 , Japan
2 Department of Island and Community Medicine, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki , Japan
3 Center for Comprehensive Community Care Education, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki , Japan
4 Department of General Medicine, Nagasaki University Hospital , Nagasaki , Japan
Background: Hepatocyte growth factor (HGF) may act as a possible biochemical index for vascular damage, although evidence for the association between HGF and carotid intima-media thickness (CIMT) is limited. Since both HGF and circulating CD34-positive cells play an important role in endothelial repair, circulating CD34-positive cell levels may influence the association between HGF and CIMT. Methods: We conducted a cross-sectional study of 269 elderly Japanese men aged 60-69 years who had undertaken an annual medical checkup from 2014 to 2015. Results: The median value for circulating CD34-positive cells was 0.93 cells/μL. Among the study population, 135 men showed low circulating CD34-positive cell levels (≤ 0.93 cells/μL). By multivariable linear regression analysis, HGF was found to be significantly positively associated with CIMT only to participants with low circulating CD34-positive cell levels, with a multi-adjusted β of 0.26 (p = 0.005) and 0.002 (0.986) for low and high circulating CD34-positive cell levels, respectively. In addition, a significant interaction was observed between HGF and circulating CD34-positive cell levels (low and high) on CIMT (multivariable p value of 0.049). A positive association exists between HGF and CIMT in elderly Japanese men, limited to participants with low circulating CD34-positive cell levels. Conclusion: A positive association exists between HGF and CIMT in community-dwelling elderly Japanese men, which is limited to participants with low numbers of circulating CD34-positive cells. Our findings indicate that circulating CD34-positive cell levels could determine the influence of HGF on CIMT in elderly Japanese men.
CD34-positive cell; CIMT; Endothelial repair; HGF; Elderly men
Hepatocyte growth factor (HGF) elicits beneficial effects
of endothelial and tissue repair following endothelial
injury since it demonstrates cytoprotective and angiogenic
] and plays an important role in tissue
]. Serum HGF may act as an indicator of
vascular endothelial disturbance since HGF levels are
elevated in participants demonstrating such a state [
HGF also plays a crucial role in inducing endothelial
progenitor cell activity such as CD34-positive cell
migration and proliferation [
]. Since CD34-positive cells
have been reported to contribute to endothelial repair
, the status of circulating CD34-positive cells could
influence the effects of HGF-mediated endothelial repair.
On the other hand, as with HGF, platelets have also
been shown to play an important role in vascular
endothelial repair in conjunction with circulating
CD34positive cells [
]. Our previous study found that
platelet count is positively associated with hypertension
in participants with low, but not high, circulating
CD34positive cell levels [
]. Since hypertension and
endothelial dysfunction have a bidirectional relationship [
circulating CD34-positive cell levels may influence the
association between HGF and atherosclerosis.
To clarify these associations, we conducted a
crosssectional study of Japanese elderly men aged 60–69 years
who had taken a general health checkup from 2014 to
The total number of male residents of Goto city aged
60–69 (estimated by the National Institute of Population
and Social Security Research in March 2013) was 3264
in 2015 [
]. The study population comprised 276 male
residents aged 60–69 years from the Goto Islands
located in the western part of Japan, who underwent an
annual medical checkup in 2014 and 2015 as
recommended by the Japanese Government. Those without
data for CD34-positive cells (n = 2), HGF (n = 3), IMT
(n = 1), or blood (n = 1) were excluded from the study
population. The remaining patients, 269 men with a
mean age of 65.4 years (standard deviation (SD), 2.6;
range 60–69), were enrolled in the study.
Data collection and laboratory measurements
Trained interviewers obtained information on medical
history. Current drinker (≥ 69 g/week) and current
smoker were defined as drinker and smoker.
Body weight and height were measured with an
automatic body composition analyzer (BF-220; Tanita,
Tokyo, Japan), and body mass index (BMI; kg/m2) was
Systolic and diastolic blood pressures of the right arm
were measured after at least 5 min of rest in a sitting
position with a blood pressure measuring device
(HEM907; Omron, Kyoto, Japan) and recorded by a trained
Fasting blood samples were collected in a heparin
sodium tube, EDTA-2K tube, and a siliconized tube. Fresh
samples (within 24 h from drawing) from the heparin
sodium tube were used to determine the number of
CD34-positive cells. BD (Beckton Dickinson Biosciences)
Trucount™ technology, an accurate and reproducible
single platform assay cited in the International Society of
Hematotherapy and Graft Engineering (ISHAGE)
] and supported by automated software on the
BD FACSCant™ II system, was used to measure the
number of circulating CD34-positive cells.
To measure HGF, serum samples were diluted fourfold
with specific Bio-Plex sample diluents. HGF
concentration was determined using a fluorescent bead-based
immunosorbent assay on a suspension array. This method
is recommended by the International Committee for
Standardization in Hematology.
Samples from the EDTA-2K tube were used to
measure white blood cell count using an automated
procedure at SRL, Inc. (Tokyo, Japan). Serum triglycerides
(TG), serum high-density lipoprotein cholesterol (HDLc),
serum γ-glutamyltranspeptidase (γ-GTP), hemoglobin
A1c (HbA1C), and serum creatinine were measured using
standard laboratory procedures at SRL, Inc. (Tokyo,
Japan). Glomerular filtration rate (GFR) was estimated by
using an established method recently proposed by a
working group of the Japanese Chronic Kidney
Disease Initiative [
]. According to this adaptation, GFR
(ml/min/1.73 m2) = 194 × (serum creatinine (enzyme
method))−1.094 × (age)−0.287.
Measurement of carotid intima-media thickness
(CIMT) by ultrasonography of the left and right carotid
arteries was performed by an experienced vascular
technician using a LOGIQ Book XP with a 10-MHz
transducer (GE Healthcare, Milwaukee, WI, USA). Mean
values for the left and right common CIMT were
calculated using automated digital edge-detection software
(Intimascope; MediaCross, Tokyo, Japan), with the
protocol described in detail elsewhere [
is an innovative software developed for CIMT
measurement to minimize measurement errors. This software
makes it possible to recognize automatically the edges of
the internal and external membranes of the blood vessels
and also to determine automatically the distance at a
sub-pixel level (estimated to be 0.01 mm) by using a
three-dimensional polynomial measurement formula
]. The reproducibility of CIMT measurements by
means of intimascope for our part of the study
population (n = 25) was shown to be satisfactory: the respective
intra-observer variations for CIMT assessed by two
examiners were simple correlation coefficients (r) = 0.98
(p < 0.001) and r = 0.97 (p < 0.001), and the
interobserver variation was r = 0.80 (p < 0.001).
Characteristics of the study population in relation to
circulating CD34-positive cell levels were expressed as
mean ± standard deviation except for TG, γ-GTP, and
HGF. Since these three factors showed a skewed
distribution, the characteristics of the study population
were expressed as median [the first quartile, the third
quartile], followed by logarithmic transformation. The
regression model for mean values was used for calculating
A simple correlation analysis and multiple linear
regression analysis of CIMT were conducted with
relevant factors adjusted for confounding factors based
on circulating CD34-positive cell levels at a median value
(0.93 cells/μL). Alcohol consumption and smoking status
are well-known factors that affect vascular remodeling.
Since γ-GTP is recognized as a factor that is influenced by
alcohol consumption, and WBC as a factor that is
influenced by smoking status [
], we added γ-GTP and
WBC as the confounding factors to the present analysis
instead of using alcohol consumption and smoking status
directly as was done in a previous study of ours [
the multiple linear regression analysis, adjustments
were made for age, systolic blood pressure (mmHg),
BMI (kg/m2), TG (mg/dL), HDLc (mg/dL), γ-GTP
(IU/L), HbA1c (%), GFR (mL/min/1.73m2), and WBC
Since bone marrow-derived endothelial progenitor
cells such as CD34-positive cells have been reported to
play an important role in maintaining the vascular
], and the level of circulating
CD34positive cells may serve as a direct indicator of vascular
maintenance activity [
14, 15, 28, 29
], we also evaluated
the association between HGF and CIMT, stratified by
circulating CD34-positive cell levels [low (< 0.93 cells/μL)
and high (≥ 0.93 cells/μL)].
To evaluate the impact of HGF levels [median value
(301.0 pg/mL)] and circulating CD34-positive cell levels
[median value (0.93 cells/μL)] on CIMT, we also created
and investigated relevant crosstabs.
All statistical analyses were performed with SAS
system for Windows (version 9.4: SAS Inc., Cary, NC).
Values of p < 0.05 were regarded as being statistically
Among the study population, 135 participants were
categorized as having low CD34-positive cell levels
(≤ 0.93 cells/μL), and 134 participants were
categorized as having high levels (> 0.93 cells/μL).
Characteristics of the study population
Characteristics of the study population based on
circulating CD34-positive cell levels are shown in
Table 1. Participants with high CD34-positive cell
levels show significantly higher values for BMI, TG,
Association between HGF and CIMT in relation to circulating CD34-positive cell levels
Table 2 shows a simple correlation analysis of CIMT
and other variables. For the total participants, HGF
was significantly positively associated with CIMT.
This association was limited to participants with low
CD34-positive cell levels, with essentially the same
associations seen from a simple linear regression analysis
(Fig. 1). After further adjustment for possible confounding
factors, for total participants even the association became
non-significant value, for participants with low and high
CD34-positive cell levels, these associations remained
unchanged (Table 3).
Effects of modification of circulating CD34-positive cell levels on the association between HGF and CIMT
As the positive association between HGF and CIMT was
observed only in participants with low circulating
CD34positive cell levels, we tested the effect of differing
CD34-positive cell levels (low and high) on the slope
of HGF and CIMT and found significant values using
both a simple (p = 0.011) and multivariable linear
model (p = 0.049).
Mean values of carotid intima-media thickness by HGF levels and CD34-positive cell levels
Among the participants with lower circulating
CD34positive cell levels than those of the reference group with
low HGF levels, significantly higher values for CIMT
were observed for participants with high HGF levels, but
not for those with high circulating CD34-positive cell
levels. No significant differences in CIMT between
participants with high and low circulating CD34-positive
cell levels were observed for participants with either high
or low HGF levels (Table 4).
The major finding of the present study is a significant
positive association between HGF and CIMT in
community-dwelling elderly Japanese men aged 60–
69 years, limited to participants with low circulating
A previous study of 317 community participants aged
over 50 reported that those with higher levels of HGF
show significantly higher CIMT values than those with
lower values (8.2 ± 1.2 mm and 7.8 ± 1.4 mm, p < 0.01,
]. Our results showing a significant
positive association between HGF and CIMT by simple
correlation analysis in total participants are compatible
with the aforementioned study.
We also found further evidence that this positive
association is limited to participants with low circulating
CD34-positive cell levels.
Serum HGF levels were elevated in participants with a
disturbance in vascular endothelial cells, indicating that
HGF could act as an indicator of vascular endothelial
]. Since HGF has both cytoprotective
and angiogenic activities [
] and plays an important
role in tissue regeneration , HGF demonstrates
beneficial effects of endothelial and tissue repair following
vascular endothelial injury.
In addition, in the bone marrow microenvironment,
HGF is known as a polyfunctional cytokine that is
produced by human bone marrow stromal cells and
directory or indirectly promotes proliferation, adhesion,
and survival of human CD34-positive cells [
CD34positive cells have been reported to contribute to
endothelial repair [
], and HGF promotes endothelial
Values are mean ± standard deviation
aValues are median [the first quartile, the third quartile]. Regression model for mean values was used for determining p values
bLogarithmic transformation was used for evaluating p
cell differentiation and increases endothelial progenitor
cell migration and proliferation [
]. Both HGF and
CD34-positive cells may be positively associated with
vascular damage. In fact, these two factors are observed
in human atherosclerotic lesions [
However, increased numbers of circulating
CD34positive cells are associated with a decrease in the extent
of subclinical atherosclerosis in asymptomatic men [
while the number of total risk factors for carotid
atherosclerosis with high levels of HGF is significantly greater
compared to low HGF among community-dwelling
]. This paradoxical phenomenon between
HGF and circulating CD34-positive cell count on
atherosclerosis may result from a consumptive reduction of
circulating CD34-positive cells. When the endothelium
sustains damage, both HGF production and circulating
CD34-positive cells become elevated. However, if the
endothelial disturbance is severe, a large proportion of
CD34-positive cells become mature cells (CD34-negative
cells) by differentiating into foam cells and endothelial
], resulting in a low level of circulating
CD34positive cells remaining. Therefore, high HGF with low
TG, γ-GTP, and HGF are calculated in logarithm values
levels of circulating CD34-positive cells may indicate the
presence of aggressive endothelial repair leading to
atherosclerosis, while higher HGF along with high levels of
circulating CD34-positive cells may indicate the
presence of sufficient endothelial repair since no evidence of
consumptive reduction of CD34-positive cells is
observed. Furthermore, our crosstab analysis showed no
statistically significant differences in CIMT values
between high and low circulating CD34-positive cell levels,
whether in participants with low or high HGF levels.
These findings also support our hypothesis that
increased productivity and consumptive reduction may
determine the circulating CD34-positive cell count for
subjects with active vascular remodeling. This hypothesis
is based on the notion that because consumptive
reduction of CD34-positive cells followed by increased
production of these cells might constitute a strong
confounding factor for the association between CIMT
and circulating CD34-positive cells among participants
with high HGF levels, while no aggressive endothelial
All listed variables have been entered in multiple linear regression analysis. TG, γ-GTP, and HGF are calculated in logarithm values
B parameter estimate, β standardized parameter estimate
Low CD34-positive cells (≤ 0.93 cells/μL)
B β p
High CD34-positive cells (> 0.93 cells/μL)
B β p
0.70 ± 0.13
repair that would affect the progression of CIMT is
necessary for those with low HGF levels.
Circulating CD34-positive cells have also been
revealed to play an important role in vascular endothelial
repair in conjunction with platelets [
platelets are significantly positively associated with
hypertension in participants with low, but not high, circulating
CD34-positive cell levels [
]. Our present results are
supported by these studies since a vicious cycle exists
between hypertension and endothelial dysfunction
], and the different associations between
circulating CD34-positive cells and platelets and
] could be induced by consumptive reduction
of the former.
Although the sample used for our study was small, it
is the largest sample used to date for a study dealing
with circulating CD34-positive cell levels in a general
elderly population selected with strict criteria, since the
participants were restricted to men in a narrow age
range because differences in gender and age can act as
strong confounding factors on associations between
CD34-positive cells, HGF, and CIMT [
this reason, even age was slightly but significantly
positively associated with CIMT (simple correlation
coefficient (r) = 0.17 p < 0.005) in our study, while no such
associations were observed for logarithm-transformed
CD34-positive cells (r = − 0.006, p = 0.034) and HGF (r =
0.03, p = 0.646).
Potential limitations of this study warrant
consideration. Although the level of circulating CD34-positive
cells influenced on the association between HGF and
CIMT significantly, no data was available with regard to
the evaluation of endothelial function. Further analyses
that include endothelial function-related data such as
flow-mediated dilation (FMD) will be necessary.
Additionally, because this was a cross-sectional study, causal
relationships were not able to be established.
In conclusion, a positive association exists between
HGF and CIMT in community-dwelling elderly
Japanese men, which is limited to participants with
low numbers of circulating CD34-positive cells. Our
findings indicate that circulating CD34-positive cell
levels could determine the influence of HGF on
CIMT in elderly Japanese men and can be expected
to serve as an effective tool for the clarification of
the roles played by HGF and CD34-positive cells in
the progression of atherosclerosis.
BD: Beckton Dickinson Biosciences; BMI: Body mass index; CIMT: Carotid
intima-media thickness; GFR: Glomerular filtration rate; HbA1c: Hemoglobin
A1c; HDLc: High-density lipoprotein cholesterol; HGF: Hepatocyte growth
factor; ISHAGE: International Society of Hematotherapy and Graft
Engineering; TG: Triglycerides; γ-GTP: γ-Glutamyltranspeptidase
We are grateful to the staff of Goto city office for their outstanding support.
This study was supported by Grants-in-Aid for Scientific Research from the
Japan Society for the Promotion of Science (No. 15K07243, No. 17H03740,
Availability of data and materials
The datasets generated and/or analyzed during the current study are not
publicly available due to ethical consideration but are available from the
corresponding author on reasonable request.
YS designed the study and performed the statistical analyses, interpreted the
data, drafted the manuscript, and revised it. SS, JK, HY, MN, SK, SN, KI, SF,
and HK assisted with the design of the study, were involved in the data
collection, and checked the manuscript. YN and TM participated in the study
concept and checked the manuscript. TM was the general coordinator and
also designed the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All procedures performed in the studies involving human participants
were in accordance with the ethical standards of the institution research
committee and with the 1964 Helsinki Declaration and its later
amendments for comparable ethical standards. The Ethics Committee for
Human Use of Nagasaki University obtained ethical approval. This study
was approved by the Ethics Committee of Nagasaki University Graduate
School of Biomedical Sciences (project registration number 14051404).
Written consent forms were available in Japanese to ensure a
comprehensive understanding of the study objectives, and informed
consent was provided by the participants.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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