Extract
Note: Please read the complete
full text with Figures and Tables at
Vascular calcification is a common finding in many cardiovascular diseases, such as hypertension, atherosclerosis,
diabetes, chronic renal failure, aging, and arterial stenosis, and is also common after prosthetic valve
replacement[1_3]. Calcified vessels have decreased capacity for vasodilatation, and increased stiffness, and promote a form of thrombus and
atherosclerotic plaque rupture. Vascular calcification is now recognized as a marker of atherosclerotic plaque burden, as well
as a major contributor to the loss of arterial compliance and increased pulse pressure that is seen with aging, diabetes, and
renal insufficiency. It is an important risk factor for cardiovascular
disease[4,5]. However, the mechanism by which vascular
calcification causes vascular dysfunction and remodeling is
unclear[6,7].
Previously, calcification was generally considered to be a process of passive calcium deposition in the extracellular matrix
and cells. That view, however, has changed in recent years, and vascular calcification is now considered to be an active,
regulative process similar to
osteogenesis[8]. During vascular calcification, the various vascular cells, including vascular
smooth muscle cells, pericytes, fibroblasts and macrophages, transform into an osteoblast-like phenotype, which is
characterized by increased alkaline phosphatase (ALP) activity, matrix vesicle formation and overexpression of bone
morphogenetic proteins (BMP) including BMP-2 and bone matrix proteins such as osteopontin (OPN), osteonectin and
osteocalcin[5,9_11].
However, changes in the function of vascular cells with the phenotypic alteration and the pathophysiological
significance of the altered phenotype remain unclear. It is common knowledge that paracrine/autocrine factors secreted from
vascular cells contribute to circulatory homeostasis and mediate the pathogenesis of cardiovascular diseases. Recent
research has shown that paracrine/autocrine dysfunction in calcified vascular vessels plays an important role in
calcification-induced vascular damage. Vasoactive substances produced by cardiovascular tissues including
adrenomedullin[12,13],
endothelin[14], C-type natriuretic peptide, parathyroid hormone-related
peptide[15], growth
factor[16], cytokine[17] and the
gaseous transmitters nitric oxide (NO) and carbon monoxide (CO) are involved in the pathophysiological process of vascular
calcification[18,19].
It is well known that gaseous transmitters such as NO and CO participate in the regulation of the pathophysiological
process of cardiovascular disease, an important target of therapeutic drugs for cardiovascular disease. Our previous research
showed that the L-arginine/NO synthase/NO/cGMP and heme/heme oxygenase/CO/cGMP pathways were altered during
vascular calcification, which suggests that NO and CO play important roles in the pathogenesis of vascular
calcification[18,19]. Endogenous hydrogen sulfide
(H2S) is a newly discovered gaseous transmitter. Two pyridoxal-5-phosphate-dependent
enzymes, cystathionine b-synthase (CBS; EC 4.2.1.22) and cystathionine
g-lyase (CSE; EC 4.4.1.1), are responsible for most
of the endogenous production of H2S in mammalian tissues that use
L-cysteine as the main
substrate[20]. Cardiovascular tissues rich in CSE, which are an important source of endogenous
H2S and have been shown to have vasodilatory, hypotensive, and
negative inotropic and growth-regulating properties, contribute to vascular
homeostasis[21_25]. However, the
pathophysiological significance of the endogenous
CSE/H2S pathway in vascular calcification is unclear. In this work, we developed a
rat vascular calcification model induced by vitamin D3 plus nicotine (VDN) to observe alterations in the vascular
CSE/H2S pathway and the effects of treatment with
H2S on vascular calcification, to explore the significance of endogenous
H2S in the pathogenesis of vascular calcification.
Materials and methods Materials All animal care and experimental
protocols complied with the animal management guidelines of the Chinese
Ministry of Health (document 55, 2001) and the Animal Care Committee of the First Hospital, Peking University. Male
Sprague-Dawley rats (weight 212±2 g) were obtained from the Animal Center, Health Science Center, Peking University
(Beijing, China). NaHS, L-cysteine, pyridoxal-5¡¯-phosphate, and vitamin D3 plus nicotine were purchased from Sigma (St
Louis, MO, USA). Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) and Trizol were obtained from Gibco (Rockville, MD, USA).
45CaCl2 was obtained from NEN Life Science (Boston, MA, USA). Specific primers for the amplification of OPN were: sense
(OPN-S) 5¡¯-CTC GCG GTG AAA GTG GCT GA-3¡¯, and antisense (OPN-A) 5¡¯-GAC CTC AGA AGA TGA ACT CT-3¡¯. Primers for
the amplification of CSE were: sense (CSE-S) 5¡¯-TCC GGA TGG AGA AAC ACT TC-3¡¯, and anti-sense (CSE-A) 5¡¯-GCT GCC
TTT AAA GCT TGA CC-3¡¯; and those for the amplification of
b-actin (for calibrating sample loading) were:
sense (b-actin-S) 5¡¯-ATC TGG CAC CAC ACC TTC-3¡¯, and antisense
(b-actin-A) 5¡¯-AGC CAG GTC CAG ACG CA-3¡¯. These oligonucleotide
primers were synthesized by SBS (Sai Bai Sheng, Beijing, China). Other chemicals and reagents were of analytical grade.
Preparation of rat vascular calcification
model We used a model version of the protocol originally described by
Niederhoffer et al[26]. Rats in the VDN group received vitamin D3 (300 000 IU/kg, im) and nicotine (25 mg/kg, orally) at 9:00 on
d 1. Nicotine administration was repeated at 19:00. Rats in the control group received an injection of normal saline (im) and two
gavages of vehicle. The rats treated with VDN were intraperitoneally injected with 2.8 or 14
mmol/kg per d of freshly prepared NaHS
(H2S donor) for 4 weeks, corresponding to low-dose and high-dose NaHS groups, respectively. All rats were housed
under standard conditions (room temperature
20±1°C, humidity 60%±10%, light from 6:00 to 18:00) and given standard
rodent chow and water freely.
At the end of week 4, the rats were anesthetized with sodium pentobarbital (45 mg/kg, ip), and catheters filled with heparin
saline (500 U/mL) were inserted into the right
femoral and right carotid arteries for measuring arterial pressure and
intraventricular pressure, respectively. A blood sample was drawn and mixed with 1 mg/mL ethylenediamine tetraacetic
acid-Na2 and 500 KIU/mL of aprotinin. Plasma was obtained by centrifugation at
1600×g for 15 min at 4°C and stored at
-70°C. The intact aortas were harvested, weighed and stored at -70 °C until use.
Measurement of calcium content in aorta Calcium content in the aorta was determined as described
previously[13]. The aortas (~10 mg) were dissolved in
HNO3 and diluted with a blank solution (27 nmol/L KCl, 27
mmol/L LaCl3). The calcium content was measured on an atomic absorption spectrophotometer at 422.7 nm (novAA 300; Analytik Jena AG, Germany).
Calcification assay (45Ca accumulation)
As described in a previous
paper[13], aortic tissue (~20 mg) was sliced and
incubated in 1 mL Krebs-Henseleit (K-H) solution (in mmol/L:
118 NaCl, 4.7 KCl, 1.3 CaCl2, 1.2
KH2PO4, 1.2 MgSO4, 25
NaHCO3, and 5 glucose; pH 7.2) with 37 kBq/mL of
45CaCl2. The reaction was stopped by the addition of ice-cold K-H solution. The
tissue was dissolved and protein content was determined by using Bradford¡¯s
method[4]. 45Ca2+ radioactivity was measured
by b-scintillation counting (LS 6500; Beckman).
Measurement of ALP activity in aortas
Tissue ALP activity was measured as described
previously[13]. An aortic homogenate was prepared in homogenizing buffer [20
mmol/L N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid (HEPES), pH 7.4,
containing 0.2% NP-40, and 20 mmol/L
MgCl2] with a Polytron (Tekmar Company, Germany) homo-genizer. After
centrifugation at 8000×g for 10 min, the supernatant was collected. The protein content of the tissue supernatant was determined by
Bradford¡¯s method[4]. An ALP activity assay was performed by mixing 200 µg of protein sample (in 200 µL) with 1 mL reaction
mixture (alkaline buffer:stock substrate solution, 1:1) with a modification of the ALP assay kit from Sigma. This mixture was
then incubated for 30 min at 37 °C. Yellow color was indicative of ALP activity.
The reaction was stopped by the addition of 12 µL of 1
mol/L NaOH, and absorbance was determined at 405 nm. ALP activity was calculated with
r-nitrophenol used as a standard, according to the manufacturer of the kit¡¯s instructions (Sigma). One unit was defined as the activity producing l
nmol of r-nitrophenol for 30 min.
Measurement of OPN and CSE mRNA in
aortas The concentration of OPN and CSE mRNA was determined by reverse
transcription-polymerase chain reaction
(RT-PCR)[27]. Total aortic RNA was prepared by
in situ lysis with Trizol reagent. One microgram of total tissue RNA was reverse-transcribed into single strand cDNA with M-MuLV reverse transcriptase and
oligo (dT) 15 primers. PCR was performed in a 0.2 mL tube containing 2 µL tissue cDNA, 5 µmol/L of each of the OPN-S and
OPN-A primers (1 µL), 2.5 mmol/L of each dNTP (1 µL), 1.5 mmol/L
MgCl2 (1.5 µL), 10× PCR buffer (2.5 µL), and 1.25 units of
Taq DNA polymerase, in a total volume of 25 µL. After being denatured at 95 °C for 5 min, the solution underwent PCR at 94
°C for 30 s, 60 °C for 30 s, and 72 °C for 40 s for 28 cycles, and then 72 °C for 5 min. Seven microliters of PCR product was
separated on a 1.5% agarose gel and stained with ethidium bromide. The optical density of the 871 bp band was measured by
use of the Gel Documentation System (Bio-Rad, Hercules, CA, USA). Amplification of OPN cDNA was confirmed with
analysis with an OPN primer measuring ribonucleotide sequence. Two microliters of PCR product was amplified again with
the 2 rat b-actin primers at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s for 23 cycles, and then at 72 °C for 5 min, and the
optical density of the b-actin band (291 bp) was measured. The relative amount of CSE mRNA (400 bp) was determined
according to the method described earlier. The solution underwent PCR at 94 °C for 30 s, 55 °C for 30 s, and 72
°C for 40 s for 30 cycles.
Measurement of CSE activity and
H2S content in plasma and aortas Tissue CSE activity was measured as described
previously[21], with minor modifications. Briefly, aortic tissue homogenate was suspended in 50 mmol/L ice-cold potassium
phosphate buffer (pH 6.8). The reaction mixture contained (in mmol/L): 100 potassium phosphate buffer (pH 7.4), 10
L-cysteine, 2 pyridoxal-5¡¯-phosphate, and 10% (w/v) tissue homogenate. Cryovial test tubes (2 mL) were used as the center
wells; each contained 0.5 mL of 1% zinc acetate as a trapping solution and a filter paper of 2.0 cm×2.5 cm to increase the
air/liquid contact surface. The reaction was performed in a 25 mL Erlenmeyer Pyrex flask. The flasks containing the reaction
mixture and the center wells were flushed with
N2 before being sealed with a double layer of Parafilm. The reaction was
initiated by transferring the flasks from ice to a shaken water bath at 37 °C . After incubation at 37 °C for 90 min, 0.5 mL of 50%
trichloroacetic acid was added to stop the reaction. The flasks were sealed again and incubated at 37 °C for another 60 min to
ensure complete trapping of the H2S released from the mixture. The contents of the center wells were then transferred to test
tubes, each containing
3.5 mL water. Subsequently, 0.5 mL of 20 mmol/L
N,N-dimethyl-p-phenylenediamine sulfate in 7.2 mmol/L HCl was added,
immediately followed by 0.4 mL of 30 mmol/L
FeCl3 in 1.2 mol/L HCl. The absorbance of the resulting solution at 670 nm was
measured using a spectrophotometer (DU 640; Beckman). The
H2S concentration was calculated using the calibration curve
of a standard H2S solution.
The tissue and plasma concentrations of
H2S were measured by using the method described earlier, without adding
l-cysteine and pyridoxal-5¡¯-phosphate to the reaction mixture.
Trichloroacetic acid was added directly into the tissue homogenates and incubated for 60 min; the plasma was then
centrifuged and the suspension collected. After adding display fluid to the suspension, optical density was measured at 670 nm.
H2S concentration was calculated by using the calibration curve.
von Kossa staining von Kossa staining for aorta was performed according to the method described by Zhang
et al[12] . A 1-cm segment of aortic arch was excised and fixed with 10% formalin. Samples were dehydrated and embedded in paraffin.
Six-micrometer-thick sections were cut, and some of the slides were stained with hematoxylin-eosin. Other slides were treated
with 5% AgNO3 for 30 min. Specimens were then counterstained with safranine (red staining) and examined under a light
microscope.
Statistical analysis The results of aortic ALP activity and
45Ca uptake were normalized to total protein and all data were
expressed as mean±SD. For comparisons between 2 groups, the unpaired Student¡¯s
t-test was used. One-way ANOVA, followed by the Student-Newman-Keuls test for significance was used to compare the 3 groups.
P<0.05 was considered statistically significant.
Results
General characteristics of vascular calcification In rats with vascular calcification induced by VDN, systolic blood
pressure was 23% higher than the blood pressure of rats in the control group. Von Kossa staining for calcium mineral
deposits produced strong positive black/brown-staining areas among the elastic fibers of the medial layer in calcified aorta
(Figure 1). Calcium content and
45Ca2+ accumulation in the calcified aorta were significantly increased by 6.8-fold and 1.4-fold,
respectively, relative to the control (P<0.01). Aortic ALP activity was significantly increased (by 1.9-fold;
P<0.01) relative to the control (Table 1). OPN mRNA concentration was increased by 39%
(P< 0.01) relative to the control (Figure 3).
Downregulation of CSE/H2S pathway in rats with vascular calcification
Compared with the controls, the plasma and aorta
H2S content was reduced by 39% and 57% (all
P<0.01, Table 2) respectively, aortic CSE activity was decreased by 53%
(P<0.01,Table 2), and aortic CSE mRNA amount was
reduced by 76%, in rats with vascular calcification
(P<0.01; Figure 2).
Administration of NaHS ameliorated vascular
calcification Administration of NaHS (a donor of
H2S) obviously reduced blood pressure in rats with vascular calcification, compared with rats that did not receive NaHS. Treatment with low and high
doses of NaHS reduced systolic blood pressure by 38%
(P<0.05) and 30% (P<0.01), respectively (Table1). NaHS significantly
reduced the aortic calcium mineral deposits (Figure 1). The rats treated with low doses of NaHS had reduced vascular calcium
content, 45Ca2 + accumulation and ALP activity by 35%, 4% and 63%
(P<0.01), respectively, and the rats in the high-dose
NaHS group had reduced calcium
content, 45Ca2 + accumulation and ALP activity by 84%, 38% and 46 %
(P<0.01), respectively. The levels of aortic OPN mRNA were decreased by 74%
(P<0.01) in the low-dose group and by 86%
(P<0.01) in the high-dose group, compared with the rats that did not receive NaHS. No significant differences in the above parameters were found
between the NaHS-treated groups (P>0.05; Table 1, Figure 3).
Discussion
Pathological calcification of cardiovascular structures, or vascular calcification, is associated with a number of diseases,
including end stage renal disease (ESRD), diabetes and cardiovascular diseases. Calcium phosphate deposition, in the form
of bioapatite, is the hallmark of vascular calcification, and can occur in the blood vessels, myocardium, and cardiac valves.
Calcified deposits are found in distinct layers of the blood vessels and are related to the underlying pathology. Intimal
calcification occurs in atherosclerotic
lesions[1,2], whereas medial calcification (also known as Monckeberg¡¯s medial sclerosis)
is associated with vascular stiffening and the arteriosclerosis observed with aging, diabetes, and
ESRD[3]. In coronary arteries, calcification is positively correlated with atherosclerotic plaque burden,
increased risk of myocardial infarction, and plaque
instability[6,7].
Previously, calcification was generally considered to be a process of passive calcium deposition in the extracellular matrix
and cells. However, elevated calcium×phosphorus product (Ca×P) cannot fully explain the pathogenic process of
cardiovascular calcification. Growing evidence indicates that vascular calcification is an actively regulated process in which vascular
cells are transformed to an osteoblast-like phenotype, which is characterized by an increase in ALP activity, matrix vesicle
formation and overexpression of marker proteins of the osteoblast phenotype. Obviously, the disturbance of vascular
paracrine/autocrine function is an important pathogenetic cause of vascular calcification. We have reported that adrenomedullin,
c-type natriuretic peptides, and parathyroid hormone-related protein secreted from the vasculature can inhibit or delay the
pathogenesis of vascular calcification, but endothelin can facilitate or intensify its pathogenesis. Interestingly, NO and CO
are protective factors against cardiovascular calcification, and the
l-arginine/NOS/NO and heme/heme oxygenase/CO
pathways are downregulated in calcified
vessels[18,19].
H2S is the recently discovered third gaseous signaling molecule. In the central nervous system, endogenous
H2S is produced in response to neuronal excitation, and alters
hippocampal long-term potentiation (LTP), a synaptic model for
memory, increasing the sensitivity of
N-methyl-D-aspartate receptors following increased intracellular cAMP, and modulates
the hypothalamo-pituitary-adrenal axis function
in vitro and in vivo[28]. Cardiovascular tissues are rich in CSE, which
catalyzes L-cysteine to generate
H2S. H2S has been shown to have vasodilatory, hypotensive, negative inotropic and
growth-regulating properties, which contribute to physiological regulation of cardiovascular homeostasis together with NO and
CO[18,19]. The endogenous
CSE/H2S pathway participates in the pathophysiological process in cardiovascular diseases such as
hypoxia-induced pulmonary hypertension[24]
, spontaneous hypertension[25], NO-deficient
hypertension[29], septic and endotoxin shock, and myocardial
ischemia[30].
Vitamin D hypervitaminosis can induce an increase in the tissue calcium content, which is deposited mainly on the elastic
fibers. Nicotine amplifies the calcifying effects of vitamin D. VDN induces calcium overload in the arteries, media calcification,
and finally widespread cardiovascular calcification. In the present study, in rats with vascular calcification induced by VDN,
systolic blood pressure was higher than that of the control group. Areas among the elastic fibers of the medial layer in the
calcified aorta stained strongly with von Kossa stain for calcium mineral deposits (Figure 1). The calcium content and
45Ca2+ accumulation in calcified aortas were significantly increased relative to controls. Aortic ALP activity and OPN mRNA levels
were significantly increased compared with controls (Figure 3). These changes in vascular calcification were in accordance
with those noted in previous
reports[26]. Interestingly, it was found that in rats with vascular calcification, plasma and aortic
H2S content was decreased, and aortic CSE activity and CSE mRNA levels were decreased (Figure 2). These results suggest
that the CSE/H2S pathway in calcified vessels was significantly inhibited. NaHS was used as a source of
H2S for the following reasons. NaHS dissociates to
Na+ and HS in solution, then HS associates with
H+ and produces H2S. It does not matter
whether the H2S solution is prepared by bubbling
H2S gas or by dissolving NaHS. In physiological saline, approximately
one-third of the H2S exists as the undissociated form
(H2S), and the remaining two-thirds exist as HS at equilibrium with
H2S[31]. The use of NaHS enables us to define the concentrations of
H2S in solution more accurately and reproducibly than bubbling
H2S gas. The influence of <1 mmol/L sodium ions on the electrophysiological experiments was negligible, because saline solution
contains 130 mmol/L sodium ions. NaHS at the concentrations used in the present study does not alter the pH. For these
reasons, NaHS has been widely used for studies of
H2S[32_34]. Administration of NaHS obviously reduced blood pressure in
rats with vascular calcification, compared with rats with vascular calcification that did not receive NaHS. Treatment with low
and high doses of NaHS decreased systolic blood pressure. NaHS significantly decreased the aortic calcium mineral deposits
(Figure 1). The rats treated with low doses of NaHS had reduced vascular calcium
content, 45Ca2+ accumulation and ALP
activity, and a high dose of NaHS produced better effects than the low dose with respect to these indices. The amount of
aortic OPN mRNA were decreased. No significant difference was found in these parameter between the 2 NaHS-treated
groups (Table 1, Figure 3) . In the present study, we used NaHS as a donor of
H2S, because it is more stable than
H2S gas, and does not change the pH value of the
plasma[32_34]. Administration of NaHS (2.8 or 14
mmol/kg per d) obviously reduced the elevated blood pressure, calcium overload, and ALP activity in the calcified vessels. von Kossa staining showed that calcium
deposition was lessened with NaHS treatment. These results suggest that
H2S could significantly inhibit the pathogenesis of
vascular calcification.
The biological effect and signal transduction pathway of
H2S have not yet been elucidated. Unlike NO and CO, the
actions of which are mediated by a second messenger, cGMP, as a physiological regulator of cardiovascular function,
H2S is mediated mainly by
KATP channel opening[20,35]. The anti-proliferation effect of
H2S on vascular smooth muscle cells could be
via inhibition of the mitogen-activated protein kinase (MAPK)
pathway[20,36]. Mody et al reported that oxygen free radicals,
such as H2O2 and oxidized low density lipoprotein were key inducers of vascular
calcification[30]. It has been reported that
H2S increases the intracellular NADPH:NADP ratio; downregulates some electron trans-porters, ATP-generating genes (including
mitochondrial cytochrome oxidase subunits I, II, III, mitochondrial cytochrome C oxidase subunit IV, and ATP synthase
subunit d), redox homeostasis genes (including glutathionine S-transferase subunit 8, glutathionine S-transferase M5,
metallothionein-2, and metallothionein-1); and decreases the redox environment in IEC-18
cells[37]. H2S might modulate
oxygen free radical release and reduce the accumulation of lipid peroxidation products.
H2S also directly clears
H2O2 and superoxide anions, antagonizes peroxynitrite-mediated damage, and it is considered to be an endogenous antagonist of
peroxyni-trite[38]. Therefore,
H2S could inhibit the development of vascular calcification by regulating oxidative stress.
In the present experiment the endogenous
CSE/H2S pathway was obviously downregulated in calcified vessels. CSE is a
key enzyme of endogenous H2S
generation in vivo[39]. But the transcriptional regulatory mechanism of CSE is still unclear.
Wang et al reported that an NO donor also enhances the expression level of CSE and increases
H2S production in cultured vascular smooth muscle
cells[29]. Our previous work showed that NO production was decreased in calcified
vessels[18]. Whether the downregulation of
CSE gene expression induced by decreased NO production in calcified vessels is
responsible for the diminished H2S generation or not needs further investigation.
The gaseous transmitters, NO, CO and
H2S, have some similar cardiovascular effects, and synergistically regulate
cardiovascular homeostasis. Our previous work showed that NO and CO are involved in the development of vascular
calcification[18,19]. In the present work, we found that
H2S also participated in the pathogenesis of vascular calcification. It has been shown
that the endogenous production of H2S
from rat aortic tissues is enhanced by NO donor
treatment[20,29]. The NO donor also enhances the expression level of
CSE in cultured vascular SMC. Hosoki et
al observed that the vasorelaxant effect of sodium
nitroprusside, an NO donor, was enhanced by incubating rat aortic tissues with
NaHS[40]. These results suggest that NO, CO
and H2S might interact with each other. Elucidation of the functions of and interactions between NO, CO and
H2S has important physiological and pathophysiological significance for understanding the pathogenic mechanisms of vascular
calcification, and could provide a new target for the prevention and treatment of vascular calcification and related diseases.
References
1 Farzaneh FA, Proudfoot D, Shanahan C, Weissberg PL. Vascular and valvar calcification: recent advances. Heart 2001; 85: 13_7.
2 Wallin R, Wajih N, Greenwood GT, Sane DC. Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy.
Med Res Rev 2001; 21: 274_301.
3 Giachelli CM. Vascular calcification
mechanisms.J Am Soc Nephrol 2004; 15: 2959_64.
4 Yang H, Curinga G, Giachelli CM. Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization
in vitro.Kidney Int 2004; 66: 2293_9.
5 Hofbauer LC, Schoppet M. Osteoprotegerin: a link between osteoporosis and arterial calcification? Lancet 2001; 358: 257_9.
6 Nandalur KR, Baskurt E, Hagspiel KD, Phillips CD, Kramer CM. Calcified carotid atherosclerotic plaque is associated less with ischemic
symptoms than is noncalcified plaque on MDCT.Am J Roentgenol 2005; 184: 295_8.
7 Schurgers LJ, Teunissen KJ, Knapen MH, Geusens P, van der Heijde D, Kwaijtaal M,
et al. Characteristics and performance of an
immunosorbent assay for human matrix Gla-protein. Clin Chim Acta 2005; 351: 131_8.
8 Basalyga DM, Simionescu DT, Xiong W, Baxter BT, Starcher BC, Vyavahare NR. Elastin degradation and calcification in an abdominal
aorta injury model: role of matrix metalloproteinases. Circulation 2004; 110: 3480_7.
9 Iba K, Takada J, Yamashita T. The serum level of bone-specific alkaline phosphatase activity is associated with aortic calcification in
osteoporosis patients.J Bone Miner Metab 2004; 22: 594_6.
10 Cola C, Almeida M, Li D, Romeo F, Mehta JL. Regulatory role of endothelium in the expression of genes affecting arterial
calcifica-tion.Biochem Biophys Res Commun 2004; 320: 424_7.
11 Abedin M, Tintut Y, Demer LL. Vascular calcification. Mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol 2004; 24:
1161_70.
12 Zhang B, Tang C, Jiang Z, Qi Y, Pang Y, Du J. Effects of adreno-medullin on vascular calcification in rats. Z Kardiol 2002; 91: 568_74.
13 Qi YF, Wang SH, Zhang BH, Bu DF, Shu TC, Du JB. Changes in amount of ADM mRNA and RAMP2 mRNA in calcified vascular smooth
muscle cells. Peptides 2003; 24: 287_94.
14 Wu SY, Zhang BH, Pan CS, Jiang HF, Pang YZ, Tang CS,
et al. Endothelin-1 is a potent
regulator in vivo in vascular calcification and in
vitro in calcification of vascular smooth muscle cells. Peptides 2003; 24: 1149_56.
15 Huang Z, Li J, Jiang Z, Qi Y, Tang C, Du J. Effects of adreno-medullin, C-type natriuretic peptide, and parathyroid hormone-related peptide
on calcification in cultured rat vascular smooth muscle cells. J Cardiovasc Pharmacol 2003; 42: 89_97.
16 Jeziorska M. Transforming growth factor-betas and CD105 expression in calcification and bone formation in human atherosclerotic
lesions. Z Kardiol 2001; 90: 23_6.
17Tintut Y, Patel J, Parhami F, Demer
LL.Tumor necrosis factor-alpha promotes
in vitro calcification of vascular cells via the cAMP
pathway. Circulation 2000; 102: 2636_42.
18 Zhang BH, Wu SY , PangYZ, Li JX, Tang CS, Du JB. Effects of
L-arginine and nitric oxide inhibitor on vascular calcification in rats. Basic
Clin Med 2003; 23: 599_603.
19 Zhang B, Wang S, Pang Y, Tang C, Du J. Alteration of heme-oxygenase-carbon monoxide pathway in calcified rat vascular smooth muscle
cells. Z Kardiol 2004; 93: 109_15.
20 Geng B, Du JB, Tang CS. Endogenous
H2S-a new gas transmitter. Prog Physiol Sci 2002; 33: 255_8.
21 Geng B, Yang J, Qi Y, Zhao J, Pang Y, Du J,
et al. H2S generated by heart in rat and its effects on cardiac function. Biochem Biophys Res
Commun 2004; 313: 362_8.
22 Du J, Hui Y, Cheung Y, Bin G, Jiang H, Chen X,
et al. The possible role of hydrogen sulfide as a smooth muscle cell proliferation inhibitor
in rat cultured cells. Heart Vessels 2004; 19: 75_80.
23 Zhong G, Chen F, Cheng Y, Tang C, Du J. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by
inhibition of nitric oxide synthase. J Hypertens 2003; 21: 1879_85.
24 Qingyou Z, Junbao D, Weijin Z, Hui Y, Chaoshu T, Chunyu Z. Impact of hydrogen sulfide on carbon monoxide/heme oxygenase pathway
in the pathogenesis of hypoxic pulmonary hyper-tension. Biochem Biophys Res Commun 2004; 317: 30_7.
25 Chen XB, Du JB, Geng B, Jiang HF, Tang CS. Changes in arterial hydrogen sulfide
(H2S) content during septic shock and endotoxin shock
in rats. Basic Clin Med 2003; 23: 384_7.
26 Niederhoffer N, Bobryshev YV, Lartaud-Idjouadiene I, Giummelly P, Atkinson J. Aortic calcification produced by vitamin D3 plus
nicotine. J Vasc Res 1997; 34: 386_98.
27 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976; 72: 248_54.
28 Qi YF, Shi YR, Bu DF, Pang YZ, Tang CS. Changes of adreno-medullin and receptor activity modifying protein 2 (RAMP2) in myocardium
and aorta in rats with isoproterenol-induced myocardial ischemia. Peptides 2003; 24: 463_8.
29 Wang R. Two's company, three's a crowd: can
H2S be the third endogenous gaseous transmitter? FASEB J 2002; 16: 1792_8.
30 Mody N, Parhami F, Sarafian TA, Demer LL. Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free
Radical Biol Med 2001; 31: 509_19.
31 Reiffenstein RJ, Hulbert WC, Roth SH. Toxicology of hydrogen sulfide. Annu Rev Pharmacol Toxicol 1992;
32:109_34.
32 Beauchamp RO Jr, Bus JS, Popp JA, Boreiko CJ, Andjelkovich DA. A critical review of the literature on hydrogen sulfide toxicity. Crit Rev
Toxicol 1984; 13:25_97.
33 Warenycia MW, Steele JA, Karpinski E, Reiffenstein RJ. Hydrogen sulfide in combination with taurine or cysteic acid reversibly abolishes
sodium currents in neuroblastoma cells. Neurotoxi-cology 1989; 10: 191_9.
34 Kombian SB, Reiffenstein RJ, Colmers WF. The actions of hydrogen sulfide on dorsal raphe serotonergic neurons
in vitro. J Neurophysiol 1993; 70:81_96.
35 Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of
H2S as a novel endogenous gaseous
KATP channel opener. EMBO J 2001; 20: 6008_16.
36 Yang G, Sun X,Wang R. Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of
mitogen-activated protein kinases and caspase-3. FASEB J 2004; 18: 1782_4.
37 Kimura Y, Kimura H. Hydrogen sulfide protects neurons from oxidative stress. FASEB J 2004; 18: 1165_7.
38 Whiteman M, Armstrong JS, Chu SH, Jia-Ling S, Wong BS, Cheung NS,
et al. The novel neuromodulator hydrogen sulfide: an
endogenous peroxynitrite ‘scavenger'? J Neurochem 2004; 90: 765_8.
39 Ishii I, Akahoshi N, Yu XN, Kobayashi Y, Namekata K, Komaki G,
et al. Murine cystathionine gamma-lyase: complete cDNA and genomic
sequences, promoter activity, tissue distribution and developmental expression. Biochem 2004; 381: 113_23.
40 Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric
oxide. Biochem Biophys Res Commun 1997; 237: 527_31.
|
