Background and aimsObesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. We reported that deficiency of Serum Amyloid A (SAA) significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. The aim of this study is to investigate whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice.MethodsMale C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with AngII until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study.ResultsPlasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas.ConclusionsWe demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

Background: Previous research has shown that the Serum Amyloid A (SAA) protein family is intricately involved in inflammatory signaling and various disease pathologies. We have previously demonstrated that SAA is associated with increased colitis disease severity and the promotion of tumorigenesis. However, the specific role of SAA proteins in breast cancer pathology remains unclear. Therefore, we investigated the role of systemic SAA1 and SAA2 (SAA1/2) in a triple-negative breast cancer mouse model. Methods: Syngeneic breast tumors were established in wild-type mice, and mice lacking the SAA1/2 (SAADKO). Subsequently, tumor volume was monitored, species survival determined, the inflammatory profiles of mice assessed with a multiplex assay, and tumor molecular biology and histology characterized with Western blotting and H&E histological staining. Results: WT tumor-bearing mice had increased levels of plasma SAA compared to wild-type control mice, while SAADKO control and tumor-bearing mice presented with lower levels of SAA in their plasma. SAADKO tumor-bearing mice also displayed significantly lower concentrations of systemic inflammatory markers. Tumors from SAADKO mice overall had lower levels of SAA compared to tumors from wild-type mice, decreased apoptosis and inflammasome signaling, and little to no tumor necrosis. Conclusions: We demonstrated that systemic SAA1/2 stimulates the activation of the NLRP3 inflammasome in breast tumors, leading to the production of pro-inflammatory cytokines. This, in turn, promoted apoptosis and tumor necrosis but did not significantly impact tumor growth or histological grading.

Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.

Serum amyloid A (SAA) is predictive of CVD in humans and causes atherosclerosis in mice. SAA has many proatherogenic effects in vitro. How-ever, HDL, the major carrier of SAA in the circula-tion, masks these effects. The remodeling of HDL by cholesteryl ester transfer protein (CETP) liberates SAA restoring its proinflammatory activity. Here, we investigated whether deficiency of SAA suppresses the previously described proatherogenic effect of CETP. ApoE-/-mice and apoE-/-mice deficient in the three acute-phase isoforms of SAA (SAA1.1, SAA2.1, and SAA3; "apoE-/-SAA-TKO") with and without adeno-associated virus-mediated expression of CETP were studied. There was no effect of CETP expression or SAA genotype on plasma lipids or in-flammatory markers. Atherosclerotic lesion area in the aortic arch of apoE-/-mice was 5.9 +/- 1.2%; CETP expression significantly increased atherosclerosis in apoE-/-mice (13.1 +/- 2.2%). However, atherosclerotic lesion area in the aortic arch of apoE-/-SAA-TKO mice (5.1 +/- 1.1%) was not significantly increased by CETP expression (6.2 +/- 0.9%). The increased athero-sclerosis in apoE-/-mice expressing CETP was associated with markedly increased SAA immuno-staining in aortic root sections. Thus, SAA aug-ments the atherogenic effects of CETP, which suggests that inhibiting CETP may be of particular benefit in patients with high SAA.

Abstract Background Past research has identified the family of Serum Amyloid A (SAA) proteins to be intricately involved in inflammatory signaling and various disease pathologies. In cancer patients, SAA proteins have been reported to be upregulated in blood and tumor tissue, correlating in many instances with disease progression. However, the exact role of SAA proteins in cancer pathology remains to be fully elucidated. We, therefore, investigated systemic SAA1 and SAA2 (SAA1/2) in modulating the inflammasome, cell cycle, apoptosis, and epithelial-to-mesenchymal transition (EMT). Methods Breast cancer allografts were established in wild-type mice (WT), and mice lacking the SAA1/2 orthologs (SAADKO). Subsequently, tumor volume was monitored, species survival determined, the inflammatory profiles of mice assessed with a multiplex assay, and tumor molecular biology and histology characterized with western blotting and H&E histological staining. Results WT tumor-bearing mice had increased levels of plasma SAA compared to WT control mice, while SAADKO control and tumor-bearing mice presented with lower levels of SAA in their plasma. SAADKO tumor-bearing mice also displayed significantly lower concentrations of the systemic inflammatory markers, IL-1β, IL-6, and IL-10 compared to WT tumor-bearing mice. Tumors from SAADKO mice overall had lower levels of SAA compared to tumors from WT mice, decreased apoptotic (cytochrome c, caspases 9/3, PARP), and inflammasome (NFκB, caspase 1, NLRP3) signaling, with little to no tumor necrosis. Conclusions We show that systemic SAA1/2 promotes the activation of the NLRP3 inflammasome in breast cancer tumors and the generation of pro-inflammatory cytokines, which promotes apoptosis and tumor necrosis. Based on these findings, we hypothesize that SAA1/2-mediated necrosis is due to NLRP3-mediated pore formation in membranes, while apoptosis signaling is likely mediated via the TIF-IA-NF-κB-apoptosis axis.

BACKGROUND:Obesity increases the risk for human abdominal aortic aneurysms (AAAs) and enhances Ang II (angiotensin II)-induced AAA formation in C57BL/6J mice. Obesity is also associated with increases in perivascular fat that expresses proinflammatory markers including SAA (serum amyloid A). We previously reported that deficiency of SAA significantly reduces Ang II-induced inflammation and AAA in hyperlipidemic apoE-deficient mice. In this study. we investigated whether adipose tissue-derived SAA plays a role in Ang II-induced AAA in obese C57BL/6J mice. METHODS:The development of AAA was compared between male C57BL/6J mice (wild type), C57BL/6J mice lacking SAA1.1, SAA2.1, and SAA3 (TKO); and TKO mice harboring a doxycycline-inducible, adipocyte-specific SAA1.1 transgene (TKO-Tgfat; SAA expressed only in fat). All mice were fed an obesogenic diet and doxycycline to induce SAA transgene expression and infused with Ang II to induce AAA. RESULTS:In response to Ang II infusion, SAA expression was significantly increased in perivascular fat of obese C57BL/6J mice. Maximal luminal diameters of the abdominal aorta were determined by ultrasound before and after Ang II infusion, which indicated a significant increase in aortic luminal diameters in wild type and TKO-TGfat mice but not in TKO mice. Adipocyte-specific SAA expression was associated with MMP (matrix metalloproteinase) activity and macrophage infiltration in abdominal aortas of Ang II-infused obese mice. CONCLUSIONS:We demonstrate for the first time that SAA deficiency protects obese C57BL/6J mice from Ang II-induced AAA. SAA expression only in adipocytes is sufficient to cause AAA in obese mice infused with Ang II.

Several studies in the past have reported positive correlations between circulating Serum amyloid A (SAA) levels and obesity. However, based on limited number of studies involving appropriate mouse models, the role of SAA in the development of obesity and obesity-related metabolic consequences has not been established. Accordingly, herein, we have examined the role of SAA in the development of obesity and its associated metabolic complications in vivo using mice deficient for all three inducible forms of SAA: SAA1.1, SAA2.1 and SAA3 (TKO). Male and female mice were rendered obese by feeding a high fat, high sucrose diet with added cholesterol (HFHSC) and control mice were fed rodent chow diet. Here, we show that the deletion of SAA does not affect diet-induced obesity, hepatic lipid metabolism or adipose tissue inflammation. However, there was a modest effect on glucose metabolism. The results of this study confirm previous findings that SAA levels are elevated in adipose tissues as well as in the circulation in diet-induced obese mice. However, the three acute phase SAAs do not play a causative role in the development of obesity or obesity-associated adipose tissue inflammation and dyslipidemia.

BACKGROUND & AIMS: Identifying new approaches to lessen inflammation, as well as the associated malignant consequences, remains crucial to improving the lives and prognosis of patients diagnosed with inflammatory bowel diseases. Although it previously has been suggested as a suitable biomarker for monitoring disease activity in patients diagnosed with Crohn's disease, the role of the acute-phase protein serum amyloid A (SAA) in inflammatory bowel disease remains unclear. In this study, we aimed to assess the role of SAA in colitis-associated cancer. METHODS: We established a model of colitis-associated cancer in wild-type and SAA double-knockout (Saa1/2(-/-)) mice by following the azoxymethane/dextran sulfate sodium protocol. Disease activity was monitored throughout the study while colon and tumor tissues were harvested for subsequent use in cytokine analyses, Western blot, and immunohistochemistry+experiments. RESULTS: We observed attenuated disease activity in mice deficient for Saa1/2 as evidenced by decreased weight loss, increased stool consistency, decreased rectal bleeding, and decreased colitis-associated tissue damage. Macrophage infiltration, including CD206(+) M2-like macrophages, also was attenuated in SAA knockout mice, while levels of interleukin 4, interleukin 10, and tumor necrosis factor-alpha were decreased in the distal colon. Mice deficient for SAA also showed a decreased tumor burden, and tumors were found to have increased apoptotic activity coupled with decreased expression for markers of proliferation. CONCLUSION: Based on these findings, we conclude that SAA has an active role in inflammatory bowel disease and that it could serve as a therapeutic target aimed at decreasing chronic inflammation and the associated risk of developing colitis-associated cancer.

Liver-derived serum amyloid A (SAA) is present in plasma where it is mainly associated with HDL and from which it is cleared more rapidly than are the other major HDL-associated apolipoproteins. Although evidence suggests that lipid-free and HDL-associated forms of SAA have different activities, the pathways by which SAA associates and disassociates with HDL are poorly understood. In this study, we investigated SAA lipidation by hepatocytes and how this lipidation relates to the formation of nascent HDL particles. We also examined hepatocyte-mediated clearance of lipid-free and HDL-associated SAA. We prepared hepatocytes from mice injected with lipopolysaccharide or an SAA-expressing adenoviral vector. Alternatively, we incubated primary hepatocytes from SAA-deficient mice with purified SAA. We analyzed conditioned media to determine the lipidation status of endogenously produced and exogenously added SAA. Examining the migration of lipidated species, we found that SAA is lipidated and forms nascent particles that are distinct from apoA-I-containing particles and that apoA-I lipidation is unaltered when SAA is overexpressed or added to the cells, indicating that SAA is not incorporated into apoA-I-containing HDL during HDL biogenesis. Like apoA-I formation, generation of SAA-containing particles was dependent on ABCA1, but not on scavenger receptor class B type I. Hepatocytes degraded significantly more SAA than apoA-I. Taken together, our results indicate that SAA’s lipidation and metabolism by the liver is independent of apoA-I and that SAA is not incorporated into HDL during HDL biogenesis.

Inbred strains of mice have been powerful tools in the analysis of serum amyloid A (SAA) synthesis and catabolism in normal host defense and in dysfunctions such as amyloidosis. Six inbred strains (C57BL, C3H, BALB/c, DBA/2, CBA, and A) constituted about 70% of the strains used in 1600 wide-ranging research studies reviewed by Festing; with the possible exception of DBA/2, these strains are also most frequently employed to study SAA. Taken together, these studies indicate that the generic term SAA is unsuitable for future studies of SAA regulation by cytokines; since SAA is not a single entity, there is a need to specifically identify the isoform being measured. Initiation and termination of the acute phase SAA response is known to involve cytokines; however, the range of stimulatory factors appears to be broader than proinflammatory cytokines, and serum and other elements such as phorbol esters have been implicated.

Background: Identifying new approaches to lessen inflammation, as well as the associated malignant consequences, remains crucial to improving the lives and prognosis of patients diagnosed with inflammatory bowel diseases (IBD).The acute-phase protein Serum Amyloid A (SAA) is implicated in various inflammation-associated pathologies and while it has been suggested as a suitable marker for monitoring disease activity in patients with Crohn's disease, its role in IBD is poorly understood.Furthermore, increased serum SAA levels have been reported for several different types of cancers, including colon cancer.However, whether the increased serum levels are an indirect consequence of the chronic inflammation associated with cancer, or whether SAA has a direct influence on tumorigenesis, is unclear.Aim: In the current study, we aimed to assess the role of SAA in colitis-associated cancer.Methods: We established a model of colitis-associated cancer in wild-type and SAA double knockout (SAADKO, knockout for Saa1 and Saa2) mice using the azoxymethane/dextran sulfate sodium protocol.Disease activity was monitored throughout the study while blood and colon tissue were harvested at euthanasia for subsequent use in cytokine analyses, western blot and immunohistochemistry experiments.Results: Decreased disease activity was observed in SAADKO mice when compared to their wild-type counterparts as indicated by decreased weight loss, increased stool consistency and decreased rectal bleeding.H&E staining of the distal colon revealed that SAADKO mice also displayed less colitis-associated tissue damage.Decreased macrophage infiltration, as identified through immunohistochemical staining with the F4/80 macrophage marker, was observed within the distal colon of SAADKO mice when compared to wild-type mice.In addition, SAADKO mice also displayed decreased Ki-67 staining in areas of dysplasia.Furthermore, a decreased tumor burden was observed in SAADKO mice and tumors were found to exhibit decreased expression of the proliferation marker MCM2, increased expression of the apoptosis marker cleaved Caspase 3, as well as decreased expression of active b-catenin, when compared to tumors isolated from wild-type mice.Conclusion: Based on these findings, we conclude that SAA has an active role in mediating colitis-associated symptoms and promotes tumorigenesis in colitis-associated cancer.Furthermore, we propose that SAA's role extends to the infiltration of macrophages, which holds additional implications for tissue inflammation and possibly inflammationassisted tumor growth.Identifying methods to target and decrease or eliminate SAA levels could improve the quality of life and prognostic outcome of patients diagnosed with IBD. Sa1109

Objectives: Serum amyloid A (SAA) is an inflammatory mediator whose concentration in plasma is increased in individuals with acute or chronic inflammation. Circulating SAA is produced and secreted largely by the liver and is present in plasma mainly associated with HDL. While accumulating evidence suggests that lipid-free, but not HDL-associated, SAA exerts pro-inflammatory effects, the pathways by which SAA acquires lipid and is incorporated into HDL are poorly understood. In this study we investigated SAA lipidation and how such lipidation relates to the formation of nascent HDL particles. Approach and results: Primary hepatocytes were prepared from C57BL/6 mice 6 hours after i.p. injection of 1 μg/g lipopolysaccharide (LPS) or 24 hours after i.v. injection of 1x10 11 particles of an adenoviral vector expressing SAA (AdSAA) to induce hepatic SAA. Alternatively, primary hepatocytes from SAA-deficient mice were incubated with 5 - 10 μg/ml purified lipid-free mouse SAA. Media was collected after 0 - 30 hours incubation, separated by non-denaturing gradient gel electrophoresis and analyzed by Western blotting to determine the lipidation status of SAA and apoA-I, the major apolipoprotein on HDLs. Based on the migration of lipidated species, both endogenously expressed and exogenously added SAA were lipidated to form similar nascent particles that were distinct in size from apoA-I-containing particles. The lipidation of SAA secreted by hepatocytes from LPS- and AdSAA-treated mice was similar, indicating that SAA lipidation is not influenced by inflammatory signaling in the liver. Moreover, the lipidation of apoA-I was not altered when SAA was over-expressed. Studies using hepatocytes from ABCA1-deficient and SR-BI-deficient mice demonstrated that the formation of SAA-containing particles was dependent on ABCA1, but not SR-BI. Conclusions: SAA is lipidated in an ABCA1-dependent manner to form nascent particles that are distinct from apoA-I-containing particles, indicating that SAA is not incorporated into HDL during HDL biogenesis. This novel finding suggests that processes subsequent to the initial lipidation of SAA and apoA-I are involved in the association of SAA to HDL, an event that may play an important role in regulating SAA function.

The liver is the most common site of metastatic disease1. Although this metastatic tropism may reflect the mechanical trapping of circulating tumour cells, liver metastasis is also dependent, at least in part, on the formation of a 'pro-metastatic' niche that supports the spread of tumour cells to the liver2,3. The mechanisms that direct the formation of this niche are poorly understood. Here we show that hepatocytes coordinate myeloid cell accumulation and fibrosis within the liver and, in doing so, increase the susceptibility of the liver to metastatic seeding and outgrowth. During early pancreatic tumorigenesis in mice, hepatocytes show activation of signal transducer and activator of transcription 3 (STAT3) signalling and increased production of serum amyloid A1 and A2 (referred to collectively as SAA). Overexpression of SAA by hepatocytes also occurs in patients with pancreatic and colorectal cancers that have metastasized to the liver, and many patients with locally advanced and metastatic disease show increases in circulating SAA. Activation of STAT3 in hepatocytes and the subsequent production of SAA depend on the release of interleukin 6 (IL-6) into the circulation by non-malignant cells. Genetic ablation or blockade of components of IL-6-STAT3-SAA signalling prevents the establishment of a pro-metastatic niche and inhibits liver metastasis. Our data identify an intercellular network underpinned by hepatocytes that forms the basis of a pro-metastatic niche in the liver, and identify new therapeutic targets.

Serum amyloid A (SAA) is a family of acute phase reactants that are elevated in chronic inflammatory conditions such as obesity and diabetes. SAA promotes atherosclerosis in mice. Although SAA is generally thought to be exclusively an HDL apolipoprotein, we and others have detected SAA on apoB-containing lipoproteins in obese/diabetic mice and humans. The goal of this study was to investigate mechanisms underlying SAA exchange between lipoprotein fractions. Obese humans with or without metabolic syndrome or type 2 diabetes were recruited. Plasma samples were collected fasting and hourly for 8h after consumption of a high fat drink. Whereas SAA was found predominantly on HDL in fasting samples, in diabetic subjects SAA shifted from HDL to VLDL and LDL in post prandial samples. Postprandial LDL and VLDL containing SAA had increased proteoglycan binding compared to fasting LDL and VLDL from the same subject. We previously reported that HDLs remodeled by CETP release lipid-free SAA. To determine if CETP facilitates SAA exchange between particles, HDL-containing SAA was incubated with SAA-free VLDL in the presence of increasing amounts of CETP. Even in the absence of CETP, 16±3% of HDL-associated SAA shifted to VLDL; in samples with CETP, 19-28% of total SAA was found on VLDL and up to 17% was lipid-poor/lipid-free. To investigate whether CETP promotes SAA exchange in vivo, SAA-deficient mice that lack CETP were injected with HDL containing SAA and then bled at 1, 3, 6 and 24 hours. Essentially all SAA was found on HDL at each time point. In contrast, in SAA-deficient mice expressing CETP by adenoviral vector, approximately 50% and 20% of SAA was associated with the VLDL and LDL fractions, respectively, 1h after injection of HDL-containing SAA. Thus, increased CETP activity in diabetes may promote the transfer of SAA from HDL to apoB-containing lipoproteins, leading to increased retention in the vasculature.

Abstract The liver is the most common site of metastasis in pancreatic ductal adenocarcinoma (PDAC). This metastatic tropism is dependent, at least in part, on the formation of a “pro-metastatic” niche that supports tumor cell seeding and colonization in the liver. However, mechanisms that direct the formation of this niche remain poorly understood. We show using the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) model of PDAC that pancreatic tumor development enhances the susceptibility of the liver to metastatic seeding by inducing recruitment of F4/80+ and Ly6G+ myeloid cells and fibrosis within the liver. 3' mRNA sequencing (QuantSeq) on RNA isolated from the liver of KPC mice versus control PC mice revealed that the liver produces a specific set of myeloid chemoattractants, particularly serum amyloid A1 and A2 (SAA1/2), early during PDAC development. In addition, gene set enrichment analysis (GSEA) on genes upregulated in the liver of KPC mice demonstrated a significant enrichment of the interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway. Consistent with this finding, phosphorylation of STAT3 was detected in 20-30% of F4/80+ myeloid cells and 80-90% of hepatocytes. A requirement for IL-6/STAT3 signaling in the formation of a pro-metastatic niche was determined by comparing the metastatic potential of wild type mice, Il-6 knockout (Il-6-/-) mice, and mice treated with anti-IL-6 receptor (IL-6R) antibody after orthotopic implantation of KPC-derived PDAC cells. Compared to wild type mice, the liver of Il-6-/- mice and mice treated with anti-IL-6R antibody was less susceptible to metastatic seeding and showed significantly less accumulation of myeloid cells, fibrosis, and production of SAA1/2 in the liver. We obtained similar results with mice that lack Stat3 specifically in hepatocytes (Stat3flox/flox Alb-Cre), demonstrating that IL-6/STAT3 signaling in hepatocytes is necessary for the formation of a pro-metastatic niche in the liver. Further, using Saa1/2 double knockout (Saa-/-) mice, we found that SAA1/2 production by hepatocytes was required for formation of the pro-metastatic niche in the liver and increased susceptibility to metastatic seeding. Patients with a history of liver metastasis also showed higher levels of SAA1/2 in the plasma compared to normal donors, and SAA overexpression was detected in hepatocytes in liver biopsy samples collected from PDAC patients. Collectively, our study reveals a novel role for hepatocytes in directing the formation of a pro-metastatic niche in the liver during PDAC development and identifies IL-6/STAT3/SAA1/2 signaling as a promising therapeutic target for prevention of metastasis in PDAC. Citation Format: Jae W. Lee, Stacy K. Thomas, Chad A. Komar, Whitney L. Gladney, Xia Hua, Dong Xin, Abraham Shaked, Mitesh J. Borad, Ramesh K. Ramanathan, Ailing Ji, Nancy R. Webb, Maria C. de Beer, Frederick C. de Beer, Paige M. Porrett, Gregory L. Beatty. IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1102.

Objective— SAA (serum amyloid A) is a family of acute-phase reactants that have proinflammatory and proatherogenic activities. SAA is more lipophilic than apoA-I (apolipoprotein A-I), and during an acute-phase response, <10% of plasma SAA is found lipid-free. In most reports, SAA is found exclusively associated with high-density lipoprotein; however, we and others have reported SAA on apoB (apolipoprotein B)–containing lipoproteins in both mice and humans. The goal of this study was to determine whether SAA is an exchangeable apolipoprotein. Approach and Results— Delipidated human SAA was incubated with SAA-free human lipoproteins; then, samples were reisolated by fast protein liquid chromatography, and SAA analyzed by ELISA and immunoblot. Both in vitro and in vivo, we show that SAA associates with any lipoprotein and does not remain in a lipid-free form. Although SAA is preferentially found on high-density lipoprotein, it can exchange between lipoproteins. In the presence of CETP (cholesterol ester transfer protein), there is greater exchange of SAA between lipoproteins. Subjects with diabetes mellitus, but not those with metabolic syndrome, showed altered SAA lipoprotein distribution postprandially. Proteoglycan-mediated lipoprotein retention is thought to be an underlying mechanism for atherosclerosis development. SAA has a proteoglycan-binding domain. Lipoproteins containing SAA had increased proteoglycan binding compared with SAA-free lipoproteins. Conclusions— Thus, SAA is an exchangeable apolipoprotein and increases apoB-containing lipoproteins’ proteoglycan binding. We and others have previously reported the presence of SAA on low-density lipoprotein in individuals with obesity, diabetes mellitus, and metabolic syndrome. We propose that the presence of SAA on apoB-containing lipoproteins may contribute to cardiovascular disease development in these populations.

Background and aims: Serum amyloid A (SAA) predicts cardiovascular events. Overexpression of SAA increases atherosclerosis development; however, deficiency of two of the murine acute phase isoforms, SAA1.1 and SAA2.1, has no effect on atherosclerosis. SAA3 is a pseudogene in humans, but is an expressed acute phase isoform in mice. The goal of this study was to determine if SAA3 affects atherosclerosis in mice. Methods: ApoE(-/-)mice were used as the model for all studies. SAA3 was overexpressed by an adenoassociated virus or suppressed using an anti-sense oligonucleotide approach. Results: Over-expression of SAA3 led to a 4-fold increase in atherosclerosis lesion area compared to control mice (p = 0.01). Suppression of SAA3 decreased atherosclerosis in mice genetically deficient in SAA1.1 and SAA2.1 (p < 0.0001). Conclusions: SAA3 augments atherosclerosis in mice. Our results resolve a previous paradox in the literature and support extensive epidemiological data that SAA is pro-atherogenic. Published by Elsevier Ireland Ltd.

Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (∼2,500-fold vs. ∼6,000-fold, respectively) and fat (∼400-fold vs. ∼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ∼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas ∼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that ∼100% of plasma SAA1.1 was recovered in HDL compared with only ∼50% of SAA2.1 and ∼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation.

Serum amyloid A (SAA) is one of the most striking acute phase reactants that can rapidly increase 1000-fold in plasma concentration in response to inflammatory cytokines. SAA in lipid-free form exhibits pro-inflammatory activities, but its putative physiological function(s) are poorly understood. SAA is produced and secreted largely by the liver and is present in plasma mainly as an HDL apolipoprotein. The pathways by which SAA is lipidated and incorporated into HDL are poorly understood. Plasma SAA is cleared more rapidly than the other major HDL apolipoproteins, but pathways involved in its delipidation and plasma clearance have also not been defined. In this study we examined how SAA is lipidated in primary hepatocytes and how such lipidation relates to the formation of nascent HDL particles. Endogenous hepatocyte SAA was lipidated and released from cells as large particles that were distinct from apoA-I-containing nascent HDL’s. Unlike apoA-I, formation of these SAA-containing particles was independent of ABCA-I. Similarly, when SAA was exogenously added to cells, SAA was lipidated to form nascent particles that were distinct from apoA-I-containing particles. We further studied the interaction of lipid-free and HDL-bound SAA with hepatocytes. Both in lipid-free form and as part of HDL, SAA exhibited significantly greater binding to cells than apoA-I or apoA-II. Binding studies were also carried out with normal and acute phase HDL’s isolated from control and SAA-deficient mice. Together, the results suggested that SAA, unlike apoA-I, is selectively removed from HDL by binding to hepatocytes. These findings may provide new insights into SAA metabolism and function.

Acute phase serum amyloid (SAA) is a family of evolutionarily conserved, secreted proteins that exerts innate functions relevant to vascular disease. In humans, two SAA isoforms (SAA1 and SAA2) are highly induced in the liver and extrahepatic tissues under the regulation of inflammatory cytokines. During severe inflammation, SAA1/2 levels can increase ≥1000-fold in plasma, where it is found associated with HDL. Mice produce an additional acute phase SAA, SAA3, which is thought to be produced mainly by adipocytes and macrophages and has not previously been found circulating on HDL. The goal of this study was to investigate whether SAA3 serves as a third liver-derived, HDL-associated acute phase SAA in mice. Using isoform-specific oligonucleotide primers for qRT-PCR, we determined that SAA3 is transcriptionally induced to a similar extent (~2500-fold) compared to SAA1.1/2.1 (~6000-fold) in livers of C57BL/6 mice 19 hr after lipopolysaccharide (LPS) injection (100 μg/mouse). SAAs were also robustly induced in fat tissue (SAA1/2~100-fold; SAA3~400-fold). The analysis of primary mouse hepatocytes and in situ hybridization of mouse liver sections indicated that liver-derived SAAs are produced by hepatocytes and not other stromal cells, including Kupffer cells. All 3 SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ~20% of SAA1/2. After separation by FPLC, virtually all of plasma SAA1/2 eluted with the HDL fraction, whereas ~15% of plasma SAA3 appeared to be lipid poor/free. HDL isolated from acute phase mouse plasma by density gradient ultracentrifugation was subjected to isoelectric focusing to determine the relative recovery of the various SAA isoforms. Whereas the bulk of plasma SAA1.1 was found in the d=1.063-1.21 fraction, only ~50% of SAA2.1 and ~10% of SAA3 was recovered after ultracentrifugation. These findings suggest that SAA3 may be more loosely associated with HDL compared to SAA1.1/2.1, which may give rise to lipid poor/free SAA3 that is susceptible to more rapid clearance in vivo. We conclude that SAA3 is a major hepatic acute phase SAA in mice that may produce systemic effects during inflammation. Future studies investigating SAA pathobiology in mice must take into account the previously under-studied SAA3.