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Introduction
Glucose and other reducing sugars react with proteins
by a series of reactions to form a class of heterogeneous,
nonenzymatic sugar-amino adducts that are called advanced
glycation endproducts (AGE)[1,2]. Numerous studies have
indicated that the formation of AGE in long-lived connective
tissue and matrix components is a causative factor in the
development of diabetic complications and diseases
associated with aging[3-6]. In the cardiovascular system, the
accumulation of AGE on structural tissue
pro teins is one of the main mechanisms underlying cardiovascular
stiffness[7,8]. Recently, a number of natural or synthetic compounds that
target AGE, including AGE inhibitors and breakers, have been
discovered and are being further
developed[9-11]. Amino-guanidine (AG) was the first compound designed to inhibit
AGE formation and cross-linking in vitro and
in vivo, and is currently undergoing phase III clinical
trials[12,13]. ALT-711, a well-known AGE breaker, has also been reported to be
effective in in vitro and animal studies, and is
currently undergoing phase II clinical
trials[14-19]. Therefore, treatment
targeting AGE is believed to be a potential effective therapeutic
option for cardiovascular
dysfunction[10].
Based on the hypothesis that AGE crosslinks could be
cleaved with N-phenacylthiazolium
bromide[20], the lead compound ALT-711, novel AGE breakers were synthesized in
our laboratory by using computer-aided drug design.
Preliminary biological screening tests demonstrated that
3-[2-(4-bromo-phenyl)-1-methyl-2-oxo-ethyl]-4,5,6,7-tetrahydro-
benzothiazol-3-ium bromide (C16; Figure 1) had the ability to
break AGE crosslinks in
vitro[21]. C16 produced a
concentration-dependent release of bovine serum albumin (BSA) from
preformed AGE-modified BSA (AGE-BSA)-collagen complexes and C16 treatment decreased the red blood cell
(RBC)-immunoglobulin G (IgG) crosslinks (unpublished data).
Therefore, the aim of the present study was to investigate
the effects of C16 on the cardiovascular system in
experimental diabetic rats. Furthermore, the action site of C16
in vivo was explored by comparison with that of ALT-711.
Materials and methods
Reagents and compounds
3-[2-(4-Bromo-phenyl)-1-methyl-2-oxo-ethyl]-4,5,6,7-tetrahydro-benzothiazol-3-ium
bromide (C16), whose structure (Figure 1) was identified by
nuclear magnetic resonance spectroscopy-mass
spectroscopy and elemental analysis, and ALT-711 were synthesized
at the Beijing Institute of Pharmacology and Toxicology, as
described previously[21]. Streptozotocin (STZ) was purchased from Sigma. AGE antibody was kindly donated by
the Beijing Institute of Radiation Medicine. All other
chemicals and substances were of analytical grade unless stated
otherwise.
Animals Diabetes was induced in 9-10-week-old male
Wistar rats by ip injection of 70 mg/kg of STZ after an
overnight fast. Only animals that developed blood glucose
levels >15 mmol/L were used. After 12 weeks of diabetes, the
animals were used for studies. For the hemodynamic study
of the left ventricle, diabetic rats were divided into 4 groups
(8 rats in each group) to assess the exact hemodynamic
changes in the left ventricle (LV) that were caused by the
diabetic state. Rats were given either vehicle or 25 mg/kg per
day of ALT-711, or 25 or 50 mg/kg per day of C16 (ig) for 4
weeks. In another hemodynamic study, 6 groups of diabetic
rats (n=8) received either vehicle or 12.5 mg/kg per day of
ALT-711, or 12.5, 25 or 50 mg/kg per day of C16 (ig) for 4
weeks, or 50 mg/kg per day of C16 (ig) for 2 weeks to assess
the possible reversal of diabetes-induced cardiovascular
abnormalities. ALT-711 and C16 were dissolved in distilled
water immediately before administration. An additional group
of age-matched nondiabetic rats served as normal controls,
and were observed in parallel for each study.
Hemodynamic study of the left ventricle Details
regarding the surgical procedure and hemodynamic measurements
have been described elsewhere[22]. In summary, animals were
anesthetized with 50 mg/kg of pentobarbital (ip). A
fluid-filled catheter was introduced through the right carotid
artery into the left ventricle. Tracings of LV pressure were
digitized at a rate of 2000 samples/s with a commercially
available analog-to-digital converter (MP150WS, BIOPAC
Systems) and a personal computer using dedicated software
(Acknowledge, Version 3, BIOPAC Systems). The digitized
LV pressure recording was used to calculate the maximal rate
of pressure rise
(+dp/dtmax) and the maximal rate of pressure
fall (-dp/dtmax).
Hemodynamic study of cardiovascular system
After animals were anesthetized with 50 mg/kg of pentobarbital (ip), a
midsternal thoracotomy was performed, and the ascending
aorta was dissected free. The pressure transducer was
advanced into the ascending aorta. An adapted Doppler probe
was positioned around the vessel to measure phasic aortic
blood flow. The system was allowed to stabilize for 10 min
before aortic blood flow and pressure were digitized at a rate
of 2000 samples/s with a commercially available
analog-to-digital converter and a personal computer using dedicated
software. All parameters were calculated on a beat-to-beat
basis for 30 s and then averaged. In steady-state conditions,
measurements were obtained of systolic and diastolic blood
pressure (SBP, DBP), cardiac output (CO), and heart rate
(HR). Total peripheral resistance (TPR) was determined as
the quotient of mean arterial blood pressure and
CO[23]. Systemic arterial compliance (SAC) was calculated from the
quotient of stroke volume and pulse
pressure[24].
RBC-IgG assay Detailed methods have been described
elsewhere[14]. Briefly, blood samples were collected, before
hemodynamic studies of the left ventricle and RBC-IgG
determinations were performed by using an anti-IgG
enzyme-linked immunosorbent assay (ELISA) adapted for use with
cellulose ester membrane-sealed 96-well microtiter plates
(Multiscreen-HA, Millipore). Heparinized blood was washed
3 times with phosphate-buffered saline (PBS), then the packed
RBC were diluted 1:250-1:500 in PBS. Membrane-containing
wells were blocked with 0.3 mL Superblock (Pierce), then
washed with 0.3 mL PBS/0.05% Tween, followed by 0.1 mL
PBS. RBC were gently vortexed and 50 μL aliquots were
pipetted into wells. Cells were then washed, and 50 μL of a
polyclonal rabbit anti-rat IgG (Sigma, diluted 1:25 000) was
added. After incubation at room temperature for 2 h, the
cells were washed 3 times with PBS, once with Tris-buffered
saline, and 0.1 mL of p-nitrophenyl phosphate substrate was
added (1 mg/mL in 0.1 mol/L diethanolamine buffer, pH 9.5).
The plates were read in a microplate reader (Bio-Rad 550) at
410 nm. The content of RBC-IgG was expressed as
OD410.
Tail tendon collagen solubility assay The solubility of
tail tendon collagen was measured by using a previously
reported method with modifications[25]. Briefly, after
performing a hemodynamic study on the left ventricle, the ratsĄŻ
tails were removed and the tail tendon was removed by gentle
pulling. The tendons were cleaned of debris and fat in 0.9%
NaCl over ice. The tendons were rolled into a ball, patted dry
on paper towels, then lyophilized. Following lyophilization,
tail tendons were stored at -70 oC in desiccated sealed
containers until use. Collagen samples (2 mg) were weighed and
digested with pepsin (5.0 μg pepsin/mg collagen in 0.5
mol/L acetic acid) for 2 h at 4 °C. After digestion, the samples were
centrifuged at 40 000×g for 60 min at 4
oC. The supernatant was collected and both the volume of the supernatant and
pellet were determined. Aliquots (500 μL) of the supernatant
and all of the pellets were acid hydrolyzed and analyzed for
their hydroxyproline content[26], which was assumed to make
up 14% of the collagen by weight. The recoverable collagen
was defined as the sum of collagen in the supernatant and
pellet after digestion and percentage solubility was defined
as the amount of collagen in the supernatant fraction in
relation to the total recoverable collagen.
Morphological study of arterial collagen
distribution After performing the hemodynamic study on the ratsĄŻ cardiovascular systems, 2 to 3 cm segments from the ratsĄŻ
descending thoracic aortas were fixed in 10% formalin in saline,
and embedded in paraffin for morphological and
immunohistochemical studies. Seven-micron sections of aorta were
stained with picrosirius red (Direct Red 80, Aldrich, in
aqueous picric acid) for 4 h. The collagen type III/I ratio for the
aortic media wall was measured by using polarizing light
microscopy (Nikon, E600POL) according to previously
published methods[27,28].
Immunohistochemistry for AGE Four-micron sections
of aorta were used for AGE staining. Briefly, the sections
were rehydrated and treated with 3%
H2O2/methanol followed by incubation in blocking buffer (Superblock, Pierce) for 20
min at room temperature. The sections were then incubated
with the anti-AGE antibody (diluted 1:100) for 2 h at room
temperature, washed in PBS, and incubated with goat
anti-rabbit IgG/horse radish peroxidase (Zymed). The staining
was visualized by reaction with diaminobenzidine tetrahydrochloride (Sino-American Biotechnology).
Statistical analysis All results are expressed
as mean±SD. Statistical analysis was performed by one-way
ANOVA analysis with SPSS. P<0.05 was considered
statistically significant.
Results
Hemodynamic study of the left ventricle Left ventricular
systolic pressure (LVSP),
+dp/dtmax, and
-dp/dtmax were decreased significantly
(P<0.01 vs normal control) in the
vehicle-treated diabetic group (Table 1). C16 treatment did not
result in significant weight or fasting blood glucose levels
changes (P>0.05 vs vehicle-treated diabetic rats). However,
treatment with C16 (25 or 50 mg/kg) for 4 weeks
resulted in a significant increase in all of these 3 parameters
(P<0.05 or P<0.01, Table 1) as compared with vehicle-treated diabetic
rats. There was no difference between C16-treated groups
(25 or 50 mg/kg) or between the C16-treated groups and the
ALT-711-treated group (25 mg/kg).
Hemodynamic study of the cardiovascular system
In comparison with the normal controls, the body weights and
HR of the vehicle-treated diabetic rats were lower
(P<0.01 and P<0.05, respectively), whereas the TPR and TPR index
were significantly higher (P<0.01). CO and SAC were
significantly lower (P<0.01 vs normal control) in vehicle-treated
diabetic rats. SBP, DBP, and HR were not significantly
different in the 6 groups of diabetic rats (Table 2). Treatment
with C16 for 4 weeks resulted in a dose-dependent
significant increase in CO and the CO index
(P<0.05 or P<0.01), a reduction in TPR and the TPR index
(P<0.05 or P<0.01), and
an increase in SAC (P<0.05 or P<0.01) as compared with the
vehicle-treated rats. After 4 weeks of treatment, similar
results were found in ALT-711-treated (12.5 mg/kg) rats. The
effects of C16 on the cardiovascular system resembled those
of ALT-711. Treatment with C16 (50 mg/kg) for 2 weeks
produced values that were slightly different from those
produced by the other treatment regimen. Although the
difference did not reach statistical significance for the majority of
the parameters, SAC was significantly increased
(P<0.01 vs vehicle group).
RBC-IgG assay The RBC-IgG content of normal control
rats was 0.21±0.01 and the content of vehicle-treated
diabetic rats was 0.61±0.04 (n=6;
P<0.01, Figure 2). Treatment with C16 (25 or 50 mg/kg) resulted in a significant reduction
of RBC-IgG content (0.51±0.06,
0.41±0.06; P<0.05, P<0.01,
respectively) in comparison with vehicle-treated diabetic rats.
ALT-711 treatment (25 mg/kg) also significantly reduced
RBC-IgG (0.48±0.07, P<0.01).
Collagen solubility assay In comparison with normal
control animals, the tail tendon collagen solubility of
the vehicle-treated animals tended to fall (from 63.9%±7.3 % to
37.7%±10.0 %, P<0.01; Figure 3). Compared with
vehicle-treated animals, collagen solubility was increased
significantly after treatment with C16 (25 mg/kg, 48.0%± 9.0%; 50
mg/kg, 56.8%±7.4%; P<0.05, P<0.01, respectively). ALT-711
treatment resulted in a significant increase in collagen
solubility (57.7%±6.2%, P<0.01).
Morphological study of arterial collagen distribution
When stained with picrosirius red, different types of
collagen in the aortic media wall could be distinguished by
polarizing light microscopy, where type I collagen appeared
yellow or yellow-red, and type III collagen appeared green
(Figure 4). The collagen type III/I ratio of the aortic media
wall tended to be greater in rats with diabetes (Figure 4B),
but C16 (50 mg/kg) and ALT-711 (12.5 mg/kg) treatment could
reverse this alteration (Figure 4C, 4D).
Immunohistochemistry for AGE In comparison with
normal control animals, the amount of AGE accumulated in the
aortic media wall of the vehicle-treated animals was increased.
But the amount of AGE was decreased by C16 (50 mg/kg)
and ALT-711 (12.5 mg/kg) treatment (Figure 5).
Discussion
Nearly a century ago, glycation was first recognized in
the food industry, and became known as the Maillard
reaction: a process in which food proteins crosslink and
become brown with age. In the 1980s, Brownlee et
al first described the harmful consequences of AGE formation on
the cardiovascular and renal systems in
humans[1,29] and diabetic
rats[13]. Recently, it has been thought more and more
likely that AGE and AGE crosslinks are linked to the
development of many age- and diabetes-related disorders through
structural modifications as well as receptor-mediated
path-ways, which activate growth factors, induce a number of
processes, and initiate inflammatory
reactions[30]. Therefore, targeting AGE, especially breaking established AGE
cross-links, was considered to be a novel and promising
therapeutic candidate for reversing AGE-related pathologic conditions.
In the present study, rats with STZ-induced diabetes of 16
weeks duration exhibited a marked increase in AGE and an
abnormal distribution of collagen type in the aorta. However,
significant decreases in hemodynamic parameters, such as
LV dp/dt, CO, and SAC, were also observed. These results
demonstrated that AGE-related changes in structure
eventually increased the stiffness of the arterial tree and myocardium,
which, in turn, resulted in functional changes.
Immunohistochemical assays revealed that C16, a potential AGE breaker,
could prevent the increase of AGE accumulation in the aortic
media wall of diabetic rats, and could reverse the increase in
the collagen type III/I ratio (prior studies have generally
shown an association between increased collagen type III
and/or the III/I ratio and the accumulation of AGE
cross-links[31,32]). Furthermore, both the diastolic function, as
indicated by -dp/dtmax, and the contractile function of LV, as
indicated by LVSP and +dp/dtmax, were restored significantly
by C16. The significant improvements in the hemodynamic
parameters could not be attributed to differences in blood
pressure, which did not change significantly during
treatment as compared with the vehicle-treated diabetic group.
Therefore, the improvements reflect intrinsic modifications
of the mechanical properties of the arterial wall. The
increase in SAC and the decrease in TPR indicates that through
treatment with C16 the stiffness of the aorta was reduced to
levels comparable to those observed in normal control rats.
Moreover, these effects seemed to be related to the duration
of the treatment, with stronger effects after 4 weeks than
2 weeks, which indicates that the effects of C16 were exerted
through the pathway of structural modification by slowly
breaking the established AGE crosslinks. The different
approaches used in the present study consistently show that
C16, a novel AGE crosslink breaker, exerted beneficial
cardiovascular actions and restored diabetes-associated
cardiovascular dysfunction in experimental diabetic rats by
reducing AGE, and that C16 has similar effects to ALT-711, the
well-known AGE breaker.
The presence of AGE crosslinks is thought to contribute
to increased insolubility and resistance of collagen to enzy
matic and chemical digestion[25], and IgG crosslinked to RBC
as a structure of AGE crosslinks is formed earlier than other
AGE crosslinks in vivo[14]. Thus, the susceptibility of
collagen to digestion by pepsin and the IgG-RBC crosslink
content have previously been used to provide 2 indexes of
protein crosslinking in
vivo[13,20,25]. In the present study, the
considerably improved solubility of collagen and decreased
content of IgG crosslinked to RBC after treatment with C16
demonstrated that C16 could reduce AGE crosslinks
in vivo.
In summary, the novel compound C16 has the ability to
break established AGE crosslinks and reduce AGE
accumulation in tissues in vivo. Furthermore, C16 can restore
diabetes-associated cardiovascular dysfunction in rats. This
provides a potential therapeutic approach for diabetes- and
aging-related cardiovascular disease.
References
1 Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end
products in tissue and the biochemical basis of diabetic
complications. N Engl J Med 1988; 318: 1315-21.
2 Kass DA. Getting better without AGE: new insights into the
diabetic heart. Circ Res 2003; 92: 704-6.
3 Brownlee M. Lilly Lecture 1993. Glycation and diabetic
complications. Diabetes 1994; 43: 836-41.
4 Singh R, Barden A, Mori T, Beilin L. Advanced glycation
end-products: a review. Diabetologia 2001; 44: 129-46.
5 Brownlee M. The pathological implications of protein glycation.
Clin Invest Med 1995; 18: 275-81.
6 Bucala R, Cerami A. Advanced glycosylation: chemistry, biology,
and implications for diabetes and aging. Adv Pharmacol 1992;
23: 1-34.
7 Sims TJ, Rasmussen LM, Oxlund H, Bailey AJ. The role of
glycation cross-links in diabetic vascular stiffening. Diabetologia
1996; 39: 946-51.
8 Avendano GF, Agarwal RK, Bashey RI, Lyons MM, Soni BJ,
Jyothirmayi GN, et al. Effects of glucose intolerance on
myocardial function and collagen-linked glycation. Diabetes 1999;
48: 1443-7.
9 Rahbar S, Figarola JL. Novel inhibitors of advanced glycation
endproducts. Arch Biochem Biophys 2003; 419: 63-79.
10 Vasan S, Foiles P, Founds H. Therapeutic potential of breakers
of advanced glycation end product-protein crosslinks. Arch
Biochem Biophys 2003; 419: 89-96.
11 Dukic-Stefanovic S, Schinzel R, Riederer P, Munch G. AGES in
brain ageing: AGE-inhibitors as neuroprotective and
anti-dementia drugs? Biogerontology 2001; 2: 19-34.
12 Li YM, Steffes M, Donnelly T, Liu C, Fuh H, Basgen J,
et al. Prevention of cardiovascular and renal pathology of aging by the
advanced glycation inhibitor aminoguanidine. Proc Natl Acad
Sci USA 1996; 93: 3902-7.
13 Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A.
Aminoguanidine prevents diabetes-induced arterial wall protein
cross-linking. Science 1986; 232: 1629-32.
14 Wolffenbuttel BH, Boulanger CM, Crijns FR, Huijberts MS,
Poitevin P, Swennen GN, et al. Breakers of advanced glycation
end products restore large artery properties in experimental
diabetes. Proc Natl Acad Sci USA 1998; 95: 4630-4.
15 Asif M, Egan J, Vasan S, Jyothirmayi GN, Masurekar MR, Lopez
S, et al. An advanced glycation endproduct cross-link breaker
can reverse age-related increases in myocardial stiffness. Proc
Natl Acad Sci USA 2000; 97: 2809-13.
16 Vaitkevicius PV, Lane M, Spurgeon H, Ingram DK, Roth GS,
Egan JJ, et al. A cross-link breaker has sustained effects on
arterial and ventricular properties in older rhesus monkeys. Proc
Natl Acad Sci USA 2001; 98: 1171-5.
17 Liu J, Masurekar MR, Vatner DE,
et al. Glycation end-product cross-link breaker reduces collagen and improves cardiac
function in aging diabetic heart. Am J Physiol Heart Circ Physiol
2003; 285: H2587-91.
18 Susic D, Varagic J, Ahn J, Frohlich ED. Cardiovascular and renal
effects of a collagen cross-link breaker (ALT 711) in adult and
aged spontaneously hypertensive rats. Am J Hypertens 2004;
17: 328-33.
19 Kass DA, Shapiro EP, Kawaguchi M, Capriotti AR, Scuteri A,
deGroof RC, et al. Improved arterial compliance by a novel
advanced glycation end-product crosslink breaker. Circulation
2001; 104: 1464-70.
20 Vasan S, Zhang X, Zhang X, Kapurniotu A, Bernhagen J, Teichberg
S, et al. An agent cleaving glucose-derived protein crosslinks
in vitro and in vivo. Nature 1996; 382: 275-8.
21 Li S, Cui H, Wang LL, inventors. New substituted penta azacyclo
salt kind compound and its use in treating protein ageing related
disease. CN patent 1534027. 2004 Oct 6.
22 Yamamoto K, Masuyama T, Sakata Y, Nishikawa N, Mano T,
Yoshida J, et al. Myocardial stiffness is determined by
ventricular fibrosis, but not by compensatory or excessive hypertrophy
in hypertensive heart. Cardiovasc Res 2002; 55: 76-82.
23 Levy BI, Duriez M, Phillipe M, Poitevin P, Michel JB. Effect of
chronic dihydropyridine (isradipine) on the large arterial walls of
spontaneously hypertensive rats. Circulation 1994; 90:
3024-33.
24 Yin FC, Guzman PA, Brin KP, et al. Effect of nitroprusside on
hydraulic vascular loads on the right and left ventricle of patients
with heart failure. Circulation 1983; 67: 1330-9.
25 Kochakian M, Manjula BN, Egan JJ. Chronic dosing with
aminoguanidine and novel advanced glycosylation end
product-formation inhibitors ameliorates cross-linking of tail tendon
collagen in STZ-induced diabetic rats. Diabetes 1996; 45:
1694-700.
26 Stegemann H, Stalder K. Determination of hydroxyproline. Clin
Chim Acta 1967; 18: 267-73.
27 Junqueira LC, Cossermelli W, Brentani R. Differential staining
of collagens type I, II and III by Sirius Red and polarization
microscopy. Arch Histol Jpn 1978; 41: 267-74.
28 Whittaker P, Kloner RA, Boughner DR, Pickering JG.
Quantitative assessment of myocardial collagen with picrosirius red
staining and circularly polarized light. Basic Res Cardiol 1994; 89:
397-410.
29 Brownlee M, Cerami A, Vlassara H. Advanced products of
nonenzymatic glycosylation and the pathogenesis of diabetic vascu
lar disease. Diabetes Metab Rev 1988; 4: 437-51.
30 Cooper ME. Importance of advanced glycation end products in
diabetes-associated cardiovascular and renal disease. Am J
Hypertens 2004; 17: 31S-8S.
31 Shimizu M, Umeda K, Sugihara N, Yoshio H, Ino H, Takeda R,
et al. Collagen remodelling in myocardia of patients with diabetes.
J Clin Pathol 1993; 46: 32-6.
32 Bruel A, Oxlund H. Changes in biomechanical properties,
composition of collagen and elastin, and advanced glycation end
products of the rat aorta in relation to age. Atherosclerosis 1996;
127: 155-65.
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