Atherosclerosis is a multifactorial vascular disease characterized by formation of inflammatory lesions. Elevated circulating acute phase proteins indicate disease risk. Serum amyloid A (SAA) is one such marker but its function remains unclear. To determine the role of SAA on aortic smooth muscle cell gene expression, a preliminary screen of a number of genes was performed and a strong up-regulation of expression of secretory phospholipase A 2 , group IIA (sPLA 2 ) was identified. The SAAinduced increase in sPLA 2 was validated by real time PCR, Western blot analysis, and enzyme activity assays. Demonstrating that SAA increased expression of sPLA 2 heteronuclear RNA and that inhibiting transcription eliminated the effect of SAA on sPLA 2 mRNA suggested that the increase was transcriptional. Transient transfections and electrophoretic mobility shift assays identified CAAT enhancer-binding protein (C/EBP) and nuclear factor B (NFB) as key regulatory sites mediating the induction of sPLA 2 . Moreover, SAA activated the inhibitor of NF-B kinase (IKK) in cultured smooth muscle cells. Previous reports showed that interleukin (IL)-1␤ up-regulates Pla2g2a gene transcription via C/EBP␤ and NFB. Interestingly, SAA activated smooth muscle cell IL-1␤ mRNA expression, however, blocking IL-1 receptors had no effect on SAA-mediated activation of sPLA 2 expression. Thus, the observed changes in sPLA 2 expression were not secondary to SAA-induced IL-1 receptor activation. The association of SAA with high density lipoprotein abrogated the SAA-induced increase in sPLA 2 expression. These data suggest that during atherogenesis, SAA can amplify the involvement of smooth muscle cells in vascular inflammation and that this can lead to deposition of sPLA 2 and subsequent local changes in lipid homeostasis.Elevated circulating acute phase proteins correlate with an increased risk for atherosclerosis (1-4). One such disease indicator is serum amyloid A (SAA) 2 (5). The SAA protein family consists of 12-14-kDa constitutive (SAA 4 ) and acute phase (SAA 1 , SAA 2 , and SAA 3 ) isoforms. During inflammation, there are large changes in liver-derived plasma levels of the acute phase isoforms, SAA 1 and SAA 2 (6, 7). The other acute phase isoform, SAA 3 , is extrahepatically inducible (8), and although the locus equivalent to SAA 3 was previously believed to be a pseudogene in humans, Larson and co-workers (9) demonstrated its expression by mammary gland epithelial cells. Proinflammatory stimuli induce SAA expression in liver; optimal expression is achieved with a combination of interleukin (IL)-1 and IL-6 (10, 11). Extrahepatic synthesis of SAA by synovial fibroblasts, macrophages, adipocytes, and smooth muscle cells has been documented (12-15). SAA is also expressed in atherosclerotic lesions (13,16,17). Although several roles have been suggested, the functions of SAA remain uncertain (7). Our laboratory demonstrated that in response to IL-1␣, cultured aortic smooth muscle cells synthesize SAA (18) leading to the hypothesis that during ather...

Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein β on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis.

Objective Intracellular cholesterol distribution impacts cell function, however processes influencing endogenous cholesterol trafficking remain largely unknown. Atherosclerosis is associated with vascular inflammation and these studies address the role of inflammatory mediators on smooth muscle cell cholesterol trafficking. Methods and Results Interestingly, in the absence of an exogenous cholesterol source, serum amyloid A increased [14C] oleic acid incorporation into cholesteryl ester in rat smooth muscle cells, suggesting endogenous cholesterol trafficking to the endoplasmic reticulum. [3H] cholesteryl ester accumulated in cells prelabeled with [3H] cholesterol, confirming that serum amyloid A mediated the movement of endogenous cholesterol. Cholesterol movement was dependent upon functional endolysosomes. The cholesterol oxidase sensitive pool of cholesterol decreased in serum amyloid A-treated cells. Furthermore, the mechanism whereby serum amyloid A induced cholesterol trafficking was determined to be via activation of expression of secretory phospholipase A2, group IIA (sPLA2) and sPLA2-dependent activation of sphingomyelinase. Interestingly, although neither tumor necrosis factor α nor interferon γ induced cholesterol trafficking, interleukin-1ß induced [14C] cholesteryl ester accumulation that was also dependent upon sPLA2 and sphingomyelinase activities. Serum amyloid A activates smooth muscle cell interleukin-1ß expression and although the interleukin-1 receptor antagonist inhibited the interleukin-1ß-induced cholesterol trafficking, it had no effect on the movement of cholesterol mediated by serum amyloid A. Conclusions These data support a role for inflammation in endogenous smooth muscle cell cholesterol trafficking from the plasma membrane to the endoplasmic reticulum.

Smooth muscle cells (SMCs) regulate vascular tone, and during chronic inflammation associated with atherosclerosis, SMCs contribute to the disease process via de‐differentiation from a contractile state. We study the impact of acute phase serum amyloid A (SAA), a cardiovascular risk marker that localizes to atherosclerotic plaques, on SMC function. The goal of this study was to define a role for SAA in SMC phenotypic expression. SMC marker expression was down‐regulated by SAA, consistent with de‐differentiation. Myocardin, a transcriptional co‐activator of SMC marker gene expression, was down‐regulated by SAA, and its overexpression rescued the SAA‐mediated repression of the smooth muscle α‐actin and smooth muscle 22 α (SM22) promoters. SAA‐mediated down‐regulation of SM22 promoter activity was also rescued by expression of the myocardin family members, myocardin related transcription factor (MRTF)‐A and MRTF‐B. It was reported that SAA is a ligand for the Toll‐like receptor (TLR)2, which has been implicated in atherosclerosis. Interestingly, FSL‐1 and Pam3CSK4, known TLR2 ligands, down‐regulated SM22 promoter activity, and the effects were rescued with myocardin overexpression. Moreover, knockdown of TLR2 using siRNA rescued the de‐differentiated phenotype induced by SAA. These data suggest that SAA and TLR2 activation promote SMC de‐differentiation characteristic of atherosclerosis.This work was supported by NIH grant HL079201.