Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular Calcification in ESRD Patients
et al. (2012) Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular
Calcification in ESRD Patients. PLoS ONE 7(10): e47217. doi:10.1371/journal.pone.0047217
Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular Calcification in ESRD Patients
Jing Liu 0
Kun Ling Ma 0
Min Gao 0
Chang Xian Wang 0
Jie Ni 0
Yang Zhang 0
Xiao Liang Zhang 0
Hong Liu 0
Yan Li Wang 0
Bi Cheng Liu 0
Alberico Catapano, University of Milan, Italy
0 1 Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine , Nanjing City, Jiangsu Province , People's Republic of China, 2 Department of Infection Management, Zhong Da Hospital, Southeast University School of Medicine , Nanjing City, Jiangsu Province , People's Republic of China
Background: Chronic inflammation plays a crucial role in the progression of vascular calcification (VC). This study was designed to investigate whether the low-density lipoprotein receptor (LDLr) pathway is involved in the progression of VC in patients with end-stage renal disease (ESRD) during inflammation. Methods and Results: Twenty-eight ESRD patients were divided into control and inflamed groups according to plasma Creactive protein (CRP) level. Surgically removed tissues from the radial arteries of patients receiving arteriovenostomy were used in the experiments. The expression of tumour necrosis factor-a (TNF-a) and monocyte chemotactic protein-1 (MCP-1) of the radial artery were increased in the inflamed group. Hematoxylin-eosin and alizarin red S staining revealed parallel increases in foam cell formation and calcium deposit formation in continuous cross-sections of radial arteries in the inflamed group compared to the control, which were closely correlated with increased LDLr, sterol regulatory element binding protein-2 (SREBP-2), bone morphogenetic proteins-2 (BMP-2), and collagen I protein expression, as shown by immunohistochemical and immunofluorescent staining. Confocal microscopy confirmed that inflammation enhanced the translocation of the SREBP cleavage-activating protein (SCAP)/SREBP-2 complex from the endoplasmic reticulum to the Golgi, thereby activating LDLr gene transcription. Inflammation increased alkaline phosphatase protein expression and reduced a-smooth muscle actin protein expression, contributing to the conversion of the vascular smooth muscle cells in calcified vessels from the fibroblastic to the osteogenic phenotype; osteogenic cells are the main cellular components involved in VC. Further analysis showed that the inflammation-induced disruption of the LDLr pathway was significantly associated with enhanced BMP-2 and collagen I expression. Conclusions: Inflammation accelerated the progression of VC in ESRD patients by disrupting the LDLr pathway, which may represent a novel mechanism involved in the progression of both VC and atherosclerosis.
Funding: This work was supported by the Natural Science Foundation of Jiangsu Province (BK2009279) and the National Natural Science Foundation of China
(Grants 81170792 and 81070571). The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Cardiovascular disease (CVD) is the leading cause of morbidity
among patients with end-stage renal disease (ESRD), accounting
for approximately 50% of deaths and 30% of hospitalisations in
this population . Annual CVD mortality is 1020 fold higher in
ESRD patients than in the general population, and this difference
is not completely explained by traditional risk factors .
Recently, more attention has been paid to vascular calcification
(VC), which induces arterial stiffness, high pulse pressure, and
cardiac valve dysfunction, contributing to ventricular hypertrophy
and heart failure [3,4]. Thus, VC results in an increased risk of
CVD mortality, especially in ESRD patients, regardless of
maintenance hemodialysis (HD) treatment status.
Vascular calcification is a complicated pathological process that
develops primarily within the intimal and medial layers of the
artery. Arterial intimal calcification (AIC) is an advanced form of
atherosclerosis (AS), driven by cellular necrosis, inflammation, and
lipid deposition manifested in a patchy, discontinuous course along
the artery. Specific risk factors for AIC in uraemia patients include
hyperphosphatemia, hypoalbuminemia, excessive calcium intake,
and HD duration. Arterial medial calcification (AMC) is observed
in the elastic lamella of the medial layer of the arteries. AMC is
closely associated with HD duration even in patients with no CVD
history at HD therapy onset. AMC is an active process that
involves the transformation of medial vascular smooth muscle cells
(VSMCs) from a fibroblastic to an osteogenic phenotype.
Normally, VSMCs have a contractile phenotype and constitutively
express proteins that inhibit mineralisation. In response to various
stimuli, however, VSMCs express and/or release several key
regulators of bone formation and bone structural associated
proteins, such as bone morphogenetic protein-2 (BMP-2), alkaline
phosphatase (ALP), and collagen I. In contrast, the expression of
proteins such as a-smooth muscle cell (a-SMA) and collagen IV is
reduced, ultimately transforming VSMCs into osteoblast-like cells
[5,6]. However, the precise mechanisms that cause the osteogenic
phenotype of VSMCs in calcified vessels are not completely clear.
Chronic systemic inflammation is a common feature in ESRD
patients , and it may be correlated with the accumulation of
pro-inflammatory compounds caused by a markedly decreased
glomerular filtration rate (GFR) . Other causes, including
malnutrition, metabolic acidosis, hyperparathyroidism, the
accumulation of advanced oxidation protein products and asymmetric
dimethyl arginine, contribute to the release of inflammatory
cytokines . Inflammation accelerates the progression of AS and
VC [10,11], which has been identified as an independent risk
factor for the morbidity and mortality of CVD in ESRD patients
It is well known that the low-density lipoprotein receptor (LDLr)
pathway is a feedback system with important roles in regulating
plasma and intracellular cholesterol homeostasis, and it is mainly
modulated by the concentration of intracellular cholesterol and the
interaction between sterol regulatory element binding protein
(SREBP) and SREBP cleavage-activating protein (SCAP).
Cholesterol deficiency enhances the translocation of SCAP from the
endoplasmic reticulum (ER) to the Golgi, where it cleaves SREBP,
thus increasing LDLr gene expression.
Our previous studies demonstrated that inflammation
accelerated the progression of AS by disrupting LDLr feedback regulation
[13,14]. The present study was performed to evaluate whether the
inflammation exacerbates the progression of VC in ESRD patients
and explore the underlying mechanisms.
Materials and Methods
All studies were approved by the Ethical Committee of
Southeast University. Each patient provided written informed
consent to the use of their tissues for research purposes.
Patient Selection and Clinical Data
We studied 28 ESRD patients from Zhong Da Hospital,
Southeast University between January 2010 and May 2011.
Patients with ESRD who were to undergo arteriovenostomy
before hemodialysis were included in the study. Patients with acute
infection, cancer, and/or chronic active hepatitis were excluded.
The included patients were divided into two groups based on
plasma C-reactive protein (CRP) levels: control (CRP,3.0 mg/l,
n = 14) and inflamed (CRP. = 3.0 mg/l, n = 14) group. Inflamed
group was defined as the patients with persistent increased plasma
levels of CRP checked at the start and the second week after
hospitalization. The patients were comprehensively monitored by
the symptoms, signs, and serum indexes, in order to detect timely
any confounding condition which may potentially affect serum
Clinical Biochemical Tests
Blood samples were assayed to determine erythrocyte
sedimentation rate (ESR), CRP, red blood cells (RBC), haemoglobin (Hb),
total protein (TP), albumin (ALB), glucose (GLU), triglyceride
(TG), total cholesterol (TC), low density lipoprotein (LDL), high
density lipoprotein (HDL), apolipoprotein A1 (Apo A1), Apo B,
lipoprotein (a), calcium (Ca), phosphate (P), and intact parathyroid
hormone (iPTH) using an automatic biochemistry analyser at the
clinical chemistry centre of the hospital.
Tissues from the radial artery were taken during radial-cephalic
anastomosis surgery. The tissue sections were rinsed with saline
and placed in 10% buffered formalin. After treatment,
representative sections of the grafts were obtained and embedded in
O.C.T. medium or paraffin.
Hematoxylin-eosin (HE) Staining
The paraffin embedded tissues were sliced and dewaxed. The
slices were dyed for 15 minutes with hematoxylin, dipped in 1%
hydrochloric alcohol, and then stained with 1% eosin for 3
minutes. The slices were sealed with resinene after they were
dehydrated to transparency. The results were observed under light
Alizarin red S Staining
The paraffin-embedded tissues were dewaxed and hydrated in
70% alcohol. After rinsing rapidly in distilled water, the slices were
dyed with alizarin red S solution for one minute. Positive results
were shown in an orange-red colour in light microscope (6200).
Paraffin-embedded sections (4 mm) were subjected to
immunohistochemical staining. After deparaffinisation, sections were
placed in citrate-buffered solution (pH 6.0) and then heated for
antigen retrieval. Endogenous peroxidase was blocked with 3%
hydrogen peroxide, and nonspecific antibody binding was blocked
with 10% goat serum. Subsequently, sections were incubated with
goat, rabbit or mouse anti-human primary antibodies against
TNF-a (Santa Cruz, USA), MCP-1 (Santa Cruz, USA), LDLr
(Abcam, UK), BMP-2 (Santa Cruz, USA), and collagen I (Abcam,
UK) overnight at 4uC, followed by incubation with biotinylated
secondary antibodies. Finally, a diaminobenzidine
tetrahydrochloride substrate was used to develop the reaction. The
results were observed under a light microscope (6200).
Semiquantitative analysis was performed by the software of Image-Pro
Plus version 5.0.
The frozen sections were fixed with 4% formalin solution and
then blocked with 5% bovine serum albumin (BSA). Subsequently,
the sections were incubated with rabbit, mouse, or goat
antihuman primary antibodies against SCAP (Abcam, UK), a-SMA
(Abcam, UK), Golgi (Invitrogen, USA), and ALP (Santa Cruz,
USA), followed by goat anti-rabbit Fluor 488, donkey anti-rabbit
Fluor 594, goat anti-mouse Fluor 594, and donkey anti-goat Fluor
488 secondary uorescent antibodies (Invitrogen, USA),
respectively. After washing, the slides were examined by laser confocal
microscopy (6100). Semi-quantitative analysis was performed by
the software of Image-Pro Plus version 5.0.
All the data were expressed as the mean 6 standard deviation
(SD) and were analysed with SPSS 13.0. Continuous variables
were compared between the two groups with the
independentsample t test (where appropriate), and correlations between
variables were analysed by Spearmans R coefficient. A difference
was considered significant if the P value was less than 0.05.
Basic Clinical Data of the Patients in the Two Groups
As shown in Table 1, there were no differences in age, body
weight, ESR, RBC, Hb, TP, ALB, lipid profiles, Ca, P, Ca6P, or
iPTH (P.0.05) between the inflamed group and the control
Local Upregulation of Inflammation in the Artery was
Consistent with Plasma CRP Level
Using immunohistochemical staining, we demonstrated that the
expression of TNF-a and MCP-1 were increased in the radial
artery in the inflamed group, which indicated that the local
inflammation in the artery was upregulated, consistent with the
observation of systemic inflammation stress (Fig. 1A, Fig. 1B).
Inflammation Induced Foam Cell Formation and Calcified
Plaque Deposition of Radial Arteries
HE staining showed that there was significant foam cell
formation in the radial arteries of the inflamed group compared
to the control (Fig. 2A, Fig. 2B). Interestingly, there was a parallel
increase in calcified plaque deposition in the radial arteries of the
inflamed group compared to controls, as evaluated by alizarin red
S staining, suggesting that a common mechanism could be
involved in both AS and VC (Fig. 2C, Fig. 2D).
Inflammation Disrupted the Feedback Regulation of the
To explore the potential mechanisms underlying this
phenomenon, we evaluated the effects of inflammation on the protein
expression of LDLr and SREBP-2 by immunohistochemical
staining in radial arteries. Inflammation significantly increased
LDLr and SREBP-2 protein expression (Fig. 3A IVI, Fig. 3B).
control (n = 14)
inflamed group (n = 14)
Moreover, the plasma CRP level was positively correlated with the
expression of the LDLr protein (Fig. 3C). Therefore, we further
investigated the effect of inflammation on the translocation of
SCAP escorting SREBP-2 from the ER to the Golgi in the radial
arteries. Confocal microscopy showed that inflammation
significantly increased SCAP translocation from the ER to the Golgi,
thereby activating LDLr gene transcription (Fig. 3D, Fig. 3E.).
Inflammation Accelerated VC by Contributing to VSMC
Conversion from the Fibroblastic to the Osteogenic
To investigate the possible mechanisms of VC in the context of
inflammation, we evaluated the effects of inflammation on the
expression of the bone formation biomarkers BMP-2 and collagen
I in the radial arteries during VC progression. As shown by
immunohistochemical staining, BMP-2 and collagen I protein
expression were significantly increased in the inflamed group
compared to the control group (Fig. 4A IVI, Fig. 4B). It is well
known that VSMC is one of the major cellular components
involved in the progression of AMC. Therefore, we evaluated the
protein expressions of ALP and a-SMA. As shown by
immunofluorescent staining, inflammation significantly increased ALP
expression and decreased a-SMA expression (Fig. 4C, Fig. 4D).
This suggests that inflammation induces VSMC conversion from
the fibroblastic to the osteogenic phenotype, thereby accelerating
the progression of AMC.
The Disruption of the LDLr Pathway was Closely
Associated with AS and VC of the Radial Arteries
Correlation analysis demonstrated that LDLr protein expression
was positively associated with BMP-2 and collagen I protein
expression (Fig. 5A and Fig. 5B). These findings, in combination
with those of our previous studies, suggest that the disruption of
the LDLr pathway under inflammatory stress may be closely
associated with the progression of AS and VC.
Dyslipidemia and chronic inflammation are common
complications in chronic kidney disease (CKD), especially ESRD. It has
been reported that dyslipidemia and inflammation act together as
partners in crime to accelerate the progression of AS and
vascular calcification in HD patients. Our previous in vivo and in
vitro studies showed that inflammation induced intracellular lipid
accumulation and foam cell formation by disrupting LDLr
feedback regulation, exacerbating the progression of AS .
The present study was designed to investigate whether the LDLr
pathway was involved in the progression of VC in HD patients
under inflammatory stress.
Using immunohistochemical staining, we found that TNF-a and
MCP-1 protein expression were increased in the arteries of the
inflamed group compared to controls, suggesting that local arterial
inflammation was also upregulated in the inflamed group.
By evaluating continuous cross-sections, we demonstrated that
there was a significant increase in foam cell formation and calcium
deposition in the inflamed group, as shown by hematoxylin eosin
and alizarin red S staining, respectively. The parallel pathological
changes in AS and VC appeared in the same area of the radial
artery, suggesting that a common pathway mediated by
inflammation and dyslipidemia may be involved in the progression
of both AS and VC.
We found that LDLr protein expression in the radial artery
tissues was increased in the inflamed group compared to the
control group and that this increase was positively associated with
Figure 1. Locally upregulated inflammation in the artery was consistent with the plasma CRP level. The local inflammation status in the
radial artery was examined by immunohistochemical staining. The positive areas were stained brown in cross-sections of radial arteries (Fig. 1, IVI,
original magnification 6200). The values of semiquantitative analysis for the positive areas were expressed as the mean 6SD from five patients in
each group (n = 14). * P,0.05 vs control (Fig. 1B).
Figure 2. Inflammation induced foam cell formation and calcified plaque deposition in the radial arteries. The lipid accumulation in the
radial arteries was checked by hematoxylin-eosin staining (Fig. 2A and Fig. 2B, original magnification 6200) Calcification was examined by alizarin red
S staining, and calcium deposits were stained orange-red (Fig. 2C and Fig. 2D, original magnification 6200).
plasma CRP level. Further analysis showed that upregulated LDLr
protein expression was mediated by increased SREBP-2 protein
expression and enhanced SCAP/SREBP-2 complex translocation.
These clinical findings were consistent with our previous in vivo and
in vitro studies, showing that the disruption of the LDLr pathway
played crucial roles in the progression of AS.
It is accepted that AMC, the main calcification mechanism in
CKD patients, is closely associated with the conversion of VSMCs
from fibroblastic to osteogenic phenotypes and the upregulation of
osteogenic programs . Although Proudfoot et al reported that
acetylated LDL stimulates human VSMC calcification by
promoting osteoblastic differentiation, little is known about the roles
of LDLr feedback regulation in modulating the phenotype
conversion of VSMCs in calcified vessels in the context of
inflammation. To investigate the possible link between the
disruption of the LDLr pathway and VC, we further evaluated
the effects of inflammation on AMC in the tissues of the radial
artery. Using tissues removed from the radial arteries of HD
patients, we demonstrated that inflammation significantly
increased the expression of the bone formation biomarker proteins
BMP-2 and collagen I during VC progression. Immunofluorescent
staining showed that inflammation significantly increased ALP
expression and decreased a-SMA expression. The correlation
analysis further showed that LDLr protein expression was
positively associated with BMP-2 and collagen I protein
expression. These observations suggest that inflammation accelerates the
progression of AMC by disrupting the LDLr pathway, which is
closely associated with the induction of VSMC phenotype
conversion. Recently, using LDL receptor deficient mice, Geng
Y et al  demonstrated that up-regulation of cholesterol
values of semiquantitative analysis for the positive areas are expressed as the mean 6SD from five patients at each group (n = 14). * P,0.05 vs control
(Fig. 3B). Correlation analysis of plasma CRP level with LDLr expression (Fig. 3C). The translocation of SCAP from the ER to Golgi was evaluated by
immunofluorescent staining. SCAP and Golgi are stained in green and red, respectively. The colocalisation of SCAP with Golgi was evaluated by laser
confocal microscopy (Fig. 3D, arrow indicates colocalisation, original magnification 6100). Overlay areas were quantified and expressed as the mean
6 SD from five patients at each group (n = 14). * P,0.05 vs control (Fig. 3E).
Figure 4. Inflammation accelerated the VC by contributing to the phenotype conversion of VSMC from the fibroblastic to the
osteogenic. The protein expressions of BMP-2 and Collagen I were checked by immunohistochemical staining. The positive areas were stained as
brown in cross-sections of radial arteries (Fig. 4A IVI, original magnification6200). The values of semiquantitative analysis for the positive areas were
expressed as the mean 6SD from five patients at each group (n = 14). * P,0.05 vs control (Fig. 4B). The co-expression of the ALP and a-SMA proteins
was checked by immunofluorescent double staining. ALP and a-SMA are stained with green and red fluorescence, respectively (Fig. 4C, original
magnification 6100). The ratio of green to red in the overlay areas were quantified and expressed as the mean6SD from five patients in each group
(n = 14). * P,0.05 vs control (Fig. 4D).
Figure 5. The disruption of the LDLr pathway was closely associated with VC of the radial arteries. Correlation analysis demonstrated
that LDLr protein expression was positively associated with BMP-2 (Fig. 5A, R = 0.782, P,0.01) and collagen I (Fig. 5B, R = 0.644, P,0.05) expression.
metabolism is essential for matrix mineralization by vascular cells,
which were further confirmed by our findings.
In summary, our findings demonstrate for the first time that
inflammation accelerates the progression of VC in HD patients by
disrupting the LDLr pathway. This may be a novel mechanism
involved in the progression of both VC and AS.
The main limitation was low number of patients and small size
of samples acquired from radial arteries in two groups, which may
limit providing more evidence with the evaluation of VC. CRP, as
a biomarker in this study for the evaluation of inflammatory status
in ESRD patients, should be understood objectively. Although it
has been widely accepted as a valuable and well recognized index
of inflammation, some studies recently have reported that CRP
partly depends upon commonly unmeasured factors (e. g. genetic
traits) [18,19]. In addition, as each patient had different
conditions, such as age, complications, treatments, serum levels
of phosphate, calcium, and intact parathyroid hormone, the data
could be not well-controlled.
Conceived and designed the experiments: KLM. Performed the
experiments: JN J. Liu Y. Zhang. Analyzed the data: KLM C.X. Wang B.C. Liu.
Contributed reagents/materials/analysis tools: KLM J. Liu. Wrote the
paper: MG H. Liu X.L. Zhang Y.L. Wang.
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