early atherogenesis is characterized by the recruitment of circulating leukocytes ( e.g. , monocytes / macrophages and t - lymphocytes etc . ) into artery wall , the formation of foam cells in the vessel intima , leading to the development of atherosclerotic lesions .
the inflammatory responses in the atherosclerotic lesions play a crucial role to promote plaque rupture and thrombosis in the late progression of atherosclerosis , leading to myocardial infarction and strokes ( libby , 2002 ) .
the rupture - prone plaque is featured by a large lipid core and accumulation of inflammatory cells including macrophages and t - lymphocytes ( mann & davies , 1996 ) . a family of matrix metalloproteinases ( mmps ) , which are proteolytic enzymes secreted by the inflammatory cells , play a pivotal role in degrading extracellular matrix and plaque rupturing ( newby , 2005 ) .
chemokines , a superfamily of chemotactic cytokines , not only cause the recruitment of leukocytes into the arterial wall , but also influence atherosclerotic plaque stability by enhancing mmps releases ( reape & groot , 1999 ; sheikine & hansson , 2004 ) , and thus play a crucial role in both the initiation and the progression of atherosclerosis .
leukotactin-1 ( lkn-1/ccl15 ) , a new member of the human cc chemokine family , is a potent chemoattractant for leukocytes , and it is implicated in inflammatory pathologies including atherosclerosis . our previous study and
others have shown that the levels of lkn-1/ccl15 expression are markedly up - regulated in macrophage - derived foam cells and in human atherosclerotic lesions ( lee et al . , 2002 ; yu et al . , 2004 ) , and moreover , the proatherogenic mediators such as oxidized lipoprotein and oxidative stress markedly upregulte lkn-1/ccl15 gene expression and protein release from the foam cells ( yu et al . , 2004 ) , indicating lkn-1/ccl15 plays a role in the initiation of atherogenesis .
however , it remains unclear whether lkn-1/ccl15 is associated with the progression of atherosclerosis . in the present study
, we investigated whether lkn-1/ccl15 can enhance mmp-9 release from macrophages and/or foam cells , which influence plaque rupture .
our data demonstrated that lkn-1/ccl15 enhanced mmp-9 protein release from human macrophages and macrophage - derived foam cells , indicating that lkn-1/ccl15 may be associated with the vulnerability of plaque rupture in the late stage of atherosclerosis .
the cells were plated at a density of 5  10 cells / ml in the medium containing phorbol myristate acetate ( pma , sigma ) at 10 m for 48 h to induce their differentiation into macrophages .
after washings twice with hbss without ca and mg , the pbmcs were then suspended in 10% fbs containing rpmi and incubated for 60 min at 37 to let the monocytes attach to the culture dish .
the attached monocytes were harvested by vigorous pipetting and plated at a density of 5  10 cells / ml in the medium containing pma at 10 m for 48 h to induce their differentiation into macrophages .
human low density lipoprotein ( ldl ) ( d = 1.006 - 1.063 ) was separated from freshly drawn normal plasma sample by sequential ultracentrifugation and extensively dialyzed at 4 against 0.15 mol / l nacl and 0.01% edta .
after edta was removed , ldl was subjected to oxidative modification by incubation with cu ( 5 m cuso4 , 16 h at 37 ) .
then cu was removed by extensive dialysis ( yu et al . , 2004 ) .
the extent of modification was assessed by the determination of electrophoretic mobility on agarose gels . in agarose gel electrophoresis ,
oxldl was stored at 4 and was used within 4 weeks of preparation . to prepare macrophage - derived foam cells ,
human thp-1 cells were plated at a density of 5  10 cells / ml in the medium containing pma at 10 m for 48 h to induce their differentiation into macrophages , and then the cells were treated with human oxidized ldl ( 25 g / ml ) for 4 days ( yu et al . , 2004 ) .
the gelatinolytic activities of mmp-9 in the conditioning culture medium were assayed by electrophoresis on 10% polyacrylamide gels containing 1 mg / ml gelatin at 4. page gels were run at 120 v , washed in 2.5% triton x-100 for 1 h , and then incubated for 16 h at 37 in activation buffer ( 50 mm tris - hcl , ph 7.5 , 10 mm cacl2 ) . after staining with coomassie blue ( 10% glacial acetic acid , 30% methanol and 1.5% coomassie brilliant blue ) for 2 - 3 h , the gel was destained with a solution of 10% glacial acetic acid , and 30% methanol without coomassie blue for 1 h. white lysis zones indicating gelatin degradation were revealed by staining with coomassie brilliant blue r-250 .
the cells were plated at a density of 5  10 cells / ml in the medium containing phorbol myristate acetate ( pma , sigma ) at 10 m for 48 h to induce their differentiation into macrophages .
after washings twice with hbss without ca and mg , the pbmcs were then suspended in 10% fbs containing rpmi and incubated for 60 min at 37 to let the monocytes attach to the culture dish .
the attached monocytes were harvested by vigorous pipetting and plated at a density of 5  10 cells / ml in the medium containing pma at 10 m for 48 h to induce their differentiation into macrophages .
human low density lipoprotein ( ldl ) ( d = 1.006 - 1.063 ) was separated from freshly drawn normal plasma sample by sequential ultracentrifugation and extensively dialyzed at 4 against 0.15 mol / l nacl and 0.01% edta .
after edta was removed , ldl was subjected to oxidative modification by incubation with cu ( 5 m cuso4 , 16 h at 37 ) .
then cu was removed by extensive dialysis ( yu et al . , 2004 ) .
the extent of modification was assessed by the determination of electrophoretic mobility on agarose gels . in agarose gel electrophoresis ,
to prepare macrophage - derived foam cells , human thp-1 cells were plated at a density of 5  10 cells / ml in the medium containing pma at 10 m for 48 h to induce their differentiation into macrophages , and then the cells were treated with human oxidized ldl ( 25 g / ml ) for 4 days ( yu et al . , 2004 ) .
the gelatinolytic activities of mmp-9 in the conditioning culture medium were assayed by electrophoresis on 10% polyacrylamide gels containing 1 mg / ml gelatin at 4. page gels were run at 120 v , washed in 2.5% triton x-100 for 1 h , and then incubated for 16 h at 37 in activation buffer ( 50 mm tris - hcl , ph 7.5 , 10 mm cacl2 ) . after staining with coomassie blue ( 10% glacial acetic acid , 30% methanol and 1.5% coomassie brilliant blue ) for 2 - 3 h , the gel was destained with a solution of 10% glacial acetic acid , and 30% methanol without coomassie blue for 1 h. white lysis zones indicating gelatin degradation were revealed by staining with coomassie brilliant blue r-250 .
the present study has demonstrated that lkn-1/ccl15 enhances mmp-9 protein release by macrophages and macrophages - derived foam cells , indicating that lkn-1/ccl15 may play a role in the plaque rupture of the atherosclerotic lesions .
macrophages , which are abundant in ruptured atherosclerotic plaques , express multiple mmps , weakening the plaque and making it rupture prone ( boyle , 2005 ) . among mmps , mmp-9 ( 92-kda gelatinase b ) , which is a prevalent form expressed by activated macrophages and foam cells , is found in human coronary atherosclerotic lesions ( galis & khatri , 2002 ; pasterkamp et al . , 2000 ) .
we found that lkn-1 significantly activated human thp-1 monocytes - derived macrophages and human pbmc - derived macrophages to release mmp-9 ( fig . 1a and 1b ) .
it has been shown that the circulating levels of lkn-1 are increased in plasma of atherosclerotic patients , and lkn-1 stimulates endothelial cells to release intracellular adhesion molecule-1 ( yu et al . ,
2004 ) . since mmp-9 is required for monocyte infiltration through endothelium from blood , lkn1/ccl15 action on macrophages to release mmp-9 may accelerate the recruitment of macrophages into the atherosclerotic lesions .
it has been shown that the expression levels of chemokine genes such as monocyte chemoattractant protein-1 ( mcp-1 ) and interleukin-8 ( il-8 ) are upregulated in human atherosclerotic lesions and they are associated with plaque rupture by activating endothelial cells and smooth muscle cells to release mmps ( kodali et al .
atherosclerotic lesions consist of mainly macrophages and lipid - laden foam cells , and thus excessive production of mmp-9 by lkn-1/ccl15 from macrophages and macrophage - derived foam cells in the lesions may accelerate the destabilization and rupture of the fibrous cap of the atherosclerotic plaque .
our data demonstrated that lkn-1 markedly enhanced the production of mmp-9 from macrophage - derived foam cells ( fig .
we have previously shown that proatherogenic mediators enhance lkn-1/ccl15 release from macrophages and macrophage - derived foam cells ( yu et al . , 2004 ) .
therefore , it can be postulated that macrophages and macrophage - derived foam cells in atherosclerotic lesions may enhance the local concentration of lkn-1 in the lesions , and lkn-1/ccl15 subsequently stimulate the macrophages / foam cells per se to secrete mmp-9 , indicating that lkn-1/ccl15 action on macrophages and foam cells to release mmp-9 may contribute to plaque destabilization in the progression of atherosclerosis . in conclusion , lkn-1/ccl15 enhanced mmp-9 protein secretion from thp-1 monocytic cells - derived macrophages , human pbmc - derived macrophages , and thp-1 macrophage - derived foam cells in a dose dependent manner . considering the association of mmp-9 in plaque rupture , our findings suggest that lkn-1/ccl15 could be associated with plaque destabilization in the progression of atherosclerosis . lkn-1/ccl15 may be a useful target for the management of atherosclerosis .
further studies are needed to elucidate the inhibitory effect of phytochemicals or food factors on lkn-1/ccl15 release from macrophages / foam cells .
 atherosclerosis is characterized by a chronic inflammatory disease , and chemokines play an important role in both initiation and progression of atherosclerosis development . 
 leukotactin-1 ( lkn-1/ccl15 ) , a new member of the human cc chemokine family , is a potent chemoattractant for leukocytes . 
 our previous study has demonstrated that lkn-1/ccl15 plays a role in the initiation of atherosclerosis , however , little is currently known whether lkn-1/ccl15 is associated with the progression of atherosclerosis . 
 matrix metalloproteinases ( mmps ) in human coronary atherosclerotic lesions play a crucial role in the progression of atherosclerosis by altering the vulnerability of plaque rupture . in the present study 
 , we examined whether lkn-1/ccl15 modulates mmp-9 release , which is a prevalent form expressed by activated macrophages and foam cells . human thp-1 monocytic cells and/or human peripheral blood monocytes ( pbmc ) were treated with phorbol myristate acetate to induce their differentiation into macrophages . 
 foam cells were prepared by the treatment of thp-1 macrophages with human oxidized ldl . 
 the macrophages and foam cells were treated with lkn-1/ccl15 , and the levels of mmp-9 release were measured by gelatin zymography . lkn-1/ccl15 
 significantly enhanced the levels of mmp-9 protein secretion from thp-1 monocytic cells - derived macrophages , human pbmc - derived macrophages , as well as macrophage - derived foam cell in a dose dependent manner . 
 our data suggest that the action of lkn-1/ccl15 on macrophages and foam cells to release mmp-9 may contribute to plaque destabilization in the progression of atherosclerosis . 