Galectin-3 gene inactivation reduces atherosclerotic lesions and adventitial inflammation in ApoE-deficient mice.

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins.

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.

The AGE inhibitor pyridoxamine inhibits lipemia and development of renal and vascular disease in Zucker obese rats.

BACKGROUND: In previous studies, pyridoxamine (PM) limited the formation of advanced glycation end products (AGEs) and development of nephropathy in streptozotocin-diabetic rats without affecting glycemic control. However, the lipid-lowering effects of PM and the correlation of plasma cholesterol and triglycerides with AGEs in skin collagen suggested that lipids might be an important source of AGEs in the diabetic rat. This study addresses the effects of hyperlipidemia on formation of advanced glycation and lipoxidation end products (AGE/ALEs) and the effects of PM on hyperlipidemia, hypertension, AGE/ALE formation, and development of nephropathy in the nondiabetic, Zucker obese rat. METHODS: Three groups of Zucker rats were studied: lean (Fa/fa), untreated fatty (fa/fa), and fa/fa treated with PM (2 g/L drinking water). Blood pressure, plasma lipids and creatinine, and urinary albumin were measured monthly. AGE/ALEs were measured in skin collagen by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Changes in wall thickness of the aorta and renal arterioles were evaluated by light microscopy. RESULTS: AGE/ALEs formation was increased two- to threefold in skin collagen of obese versus lean rats. PM inhibited the increases in AGE/ALEs in collagen, and significantly decreased the rise in plasma triglycerides, cholesterol, and creatinine, corrected hypertension and thickening of the vascular wall, and nearly normalized urinary protein and albumin excretion in Zucker fa/fa rats. CONCLUSION: Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.