Korean Circ J.  2010 Oct;40(10):520-526. 10.4070/kcj.2010.40.10.520.

Alagebrium Chloride, a Novel Advanced Glycation End-Product Cross Linkage Breaker, Inhibits Neointimal Proliferation in a Diabetic Rat Carotid Balloon Injury Model

• Kim JB
• Song BW
• Park S
• Hwang KC
• Cha BS
• Jang Y
• Lee HC
• Lee MH

• KMID: 1826141
• DOI: http://doi.org/10.4070/kcj.2010.40.10.520

Abstract

BACKGROUND AND OBJECTIVES
Vascular perturbation induced by advanced glycation end-products (AGEs) leads to progression of atherosclerosis, plaque instability, and vascular inflammation, which results in a higher risk of neointimal proliferation. Here we investigated the inhibitory effect of alagebrium chloride (ALT-711), a breaker of AGE-based cross links, on neointimal proliferation in a carotid artery balloon injury model in diabetic rats induced by streptozotocin (STZ).
MATERIALS AND METHODS
Rat aortic vascular smooth muscle cells (RASMCs) were treated with 1-100 microM of alagebrium added 24 hours before the addition of AGEs. This in vivo study was done using 8-week-old male rats that were injected intraperitoneally with 80 mg/kg STZ. Sixteen weeks later, the diabetic rats were treated with 10 mg/kg alagebrium for 4 weeks, after which carotid artery balloon injury was induced. After 4 weeks, the animals were sacrificed for histological analysis.
RESULTS
Proliferation of RASMCs was significantly inhibited in alagebrium-treated cells. Alagebrium dose-dependently inhibited AGE-mediated formation of reactive oxygen species (ROS), extracellular signal-regulated kinase phosphorylation, and cyclooxygenase-2 expression. The cellular mechanisms of AGE-induced connective tissue and extracellular matrix expression were decreased in the alagebrium-treated group. This in vivo study shows that expression of AGE receptors and neointima hyperplasia are significantly suppressed in balloon-injured rats treated with alagebrium.
CONCLUSION
Alagebrium treatment in diabetic rats significantly inhibits neointimal hyperplasia after carotid balloon injury due to its inhibition of intracellular ROS synthesis, which results in inhibition of RASMCs proliferation.

Keyword

Alagebrium; Advanced glycation end-products; Neointimal hyperplasia

MeSH Terms

Animals
Atherosclerosis
Carotid Arteries
Connective Tissue
Cyclooxygenase 2
Extracellular Matrix
Humans
Hyperplasia
Inflammation
Male
Muscle, Smooth, Vascular
Neointima
Phosphorylation
Phosphotransferases
Rats
Reactive Oxygen Species
Streptozocin
Thiazoles
Cyclooxygenase 2
Phosphotransferases
Reactive Oxygen Species
Streptozocin
Thiazoles

Figure

• 

  Fig. 1 Anti-proliferative effect of alagebrium in AGE-stimulated RASMCs. AGE promotes proliferation of RASMCs, but alagebrium dose-dependently inhibits proliferation of RASMCs (*p<0.05 vs. control; †p<0.01 vs. AGE-treated group). AGE: advanced glycation end-product, RASMCs: rat aortic vascular smooth muscle cells.

• 

  Fig. 2 Effect of AGE and alagebrium on the formation of intracellular ROS in RASMCs. AGEs dose-dependently stimulated growth of RASMCs. Alagebrium reduced the formation of reactive oxygen species (*p<0.05 vs. CTL; vs. AGE-treated group). AGE: advanced glycation end-product, ROS: reactive oxygen species, RASMCs: rat aortic vascular smooth muscle cells, DCF: 2'-7'-dichlorofluorescin, CTL: control.

• 

  Fig. 3 Effect of alagebrium on phosphorylation of ERK and mRNA level of COX-2. A: MAPK activation was seen within 30 minutes following 50 µg/mL of AGEs. Alagebrium significantly inhibited expression of COX-2 mRNA in AGE-stimulated RASMCs. B: these signaling activations were significantly inhibited by pretreatment with 1 and 10 µM of alagebrium (*p<0.05 vs. AGE-treated group). ERK: extracellular signal-regulated kinase, COX-2: cyclooxygenase-2, mRNA: messenger ribonucleic acid, MAPK: mitogen-activated protein kinase, AGE: advanced glycation end-product, RASMCs: rat aortic vascular smooth muscle cells.

• 

  Fig. 4 Effects of alagebrium on expression of CTGF and ECM in AGE-stimulated RASMCs. AGE (50 µg/mL) treatment for 24 hours markedly increased messenger ribonucleic acid expression of collagen type III (A), fibronectin (B) and CTGF (C) in RASMCs. Alagebrium (1 and 10 µM) dose-dependently abolished this effect (*p<0.05 vs. AGE-treated group, **p<0.01 vs. AGE-treated group). CTGF: connective tissue growth factor, ECM: extra-cellular matrix, AGE: advanced glycation end-products, RASMCs: rat aortic vascular smooth muscle cells.

• 

  Fig. 5 Effect of alagebrium on the expression of RAGE in balloon-injured rats. A: RAGE expression was significantly decreased by alagebrium treatment in vivo, which was confirmed by immunohistochemistry. All sections were observed by virtual microscopy (magnification: left, ×200; right, ×400). B: AGE group increased expression of messenger ribonucleic acid for RAGE. This effect was significantly attenuated by alagebrium (*p<0.05 vs. AGE-treated group). RAGE: advanced glycation end-product receptor.

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  Fig. 6 The effect of alagebrium on extracellular matrix and neointima formation at the site of balloon-injury in STZ-treated rats. A: neointima hyperplasia was decreased by alagebrium treatment in diabetic rats (H&E stain). B: extracellular matrix in STZ-treated rats treated with alagebrium was reduced to below control levels (*p<0.05 vs. Control). STZ: streptozotocin.

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