Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice.

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 augments the atherogenic effects of cholesteryl ester transfer protein.

Serum amyloid A (SAA) is predictive of CVD in humans and causes atherosclerosis in mice. SAA has many proatherogenic effects in vitro. However, HDL, the major carrier of SAA in the circulation, 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 inflammatory 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 atherosclerosis in apoE-/- mice expressing CETP was associated with markedly increased SAA immunostaining in aortic root sections. Thus, SAA augments the atherogenic effects of CETP, which suggests that inhibiting CETP may be of particular benefit in patients with high SAA.

Serum Amyloid A Promotes Inflammation-Associated Damage and Tumorigenesis in a Mouse Model of Colitis-Associated Cancer.

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-ɑ 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.

Serum amyloid A and inflammasome activation: A link to breast cancer progression?

Breast cancer is the most frequently diagnosed cancer in women globally. Although there have been many significant advances made in the diagnosis and treatment of breast cancer, numerous unresolved challenges remain, which include prevention, early diagnosis, metastasis and recurrence. The role of inflammation in cancer development is well established and is believed to be one of the leading hallmarks of cancer progression. Recently, the role of the inflammasome, a cytosolic multiprotein complex, has received attention in different cancers. By contributing to the activation of inflammatory cytokines the inflammasome intensifies the inflammatory cascade. The inflammasome can be activated through several pathways, which include the binding of pattern associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) to toll-like receptors (TLRs). Serum amyloid A (SAA), a non-specific acute-phase protein, can function as an endogenous DAMP by binding to pattern recognition receptors like TLRs on both breast cancer cells and cancer associated fibroblasts (CAFs). SAA can thus stimulate the production of IL-1ß, thereby creating a favourable inflammatory environment to support tumour growth. The aim of this review is to highlight the possible role of SAA as an endogenous DAMP in the tumour microenvironment (TME) thereby promoting breast cancer growth through the activation of the NLRP3 inflammasome.

High-density lipoprotein inhibits serum amyloid A-mediated reactive oxygen species generation and NLRP3 inflammasome activation.

Serum amyloid A (SAA) is a high-density apolipoprotein whose plasma levels can increase more than 1000-fold during a severe acute-phase inflammatory response and are more modestly elevated in chronic inflammation. SAA is thought to play important roles in innate immunity, but its biological activities have not been completely delineated. We previously reported that SAA deficiency protects mice from developing abdominal aortic aneurysms (AAAs) induced by chronic angiotensin II (AngII) infusion. Here, we report that SAA is required for AngII-induced increases in interleukin-1ß (IL-1ß), a potent proinflammatory cytokine that is tightly controlled by the Nod-like receptor protein 3 (NLRP3) inflammasome and caspase-1 and has been implicated in both human and mouse AAAs. We determined that purified SAA stimulates IL-1ß secretion in murine J774 and bone marrow-derived macrophages through a mechanism that depends on NLRP3 expression and caspase-1 activity, but is independent of P2X7 nucleotide receptor (P2X7R) activation. Inhibiting reactive oxygen species (ROS) by N-acetyl-l-cysteine or mito-TEMPO and inhibiting activation of cathepsin B by CA-074 blocked SAA-mediated inflammasome activation and IL-1ß secretion. Moreover, inhibiting cellular potassium efflux with glyburide or increasing extracellular potassium also significantly reduced SAA-mediated IL-1ß secretion. Of note, incorporating SAA into high-density lipoprotein (HDL) prior to its use in cell treatments completely abolished its ability to stimulate ROS generation and inflammasome activation. These results provide detailed insights into SAA-mediated IL-1ß production and highlight HDL's role in regulating SAA's proinflammatory effects.

Serum amyloid A3 is pro-atherogenic.

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 adeno-associated 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.

Deficiency of Endogenous Acute-Phase Serum Amyloid A Protects apoE-/- Mice From Angiotensin II-Induced Abdominal Aortic Aneurysm Formation.

OBJECTIVE: Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. APPROACH AND RESULTS: Plasma SAA increased ≈60-fold in apoE(-/-) mice 24 hours after intraperitoneal Ang II injection (100 µg/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE(-/-) mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE(-/-) mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II-infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. CONCLUSIONS: Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.

Deficiency of endogenous acute phase serum amyloid A does not affect atherosclerotic lesions in apolipoprotein E-deficient mice.

OBJECTIVE: Although elevated plasma concentrations of serum amyloid A (SAA) are associated strongly with increased risk for atherosclerotic cardiovascular disease in humans, the role of SAA in the pathogenesis of lesion formation remains obscure. Our goal was to determine the impact of SAA deficiency on atherosclerosis in hypercholesterolemic mice. APPROACH AND RESULTS: Apolipoprotein E-deficient (apoE(-/-)) mice, either wild type or deficient in both major acute phase SAA isoforms, SAA1.1 and SAA2.1, were fed a normal rodent diet for 50 weeks. Female mice, but not male apoE-/- mice deficient in SAA1.1 and SAA2.1, had a modest increase (22%; P≤0.05) in plasma cholesterol concentrations and a 53% increase in adipose mass compared with apoE-/- mice expressing SAA1.1 and SAA2.1 that did not affect the plasma cytokine levels or the expression of adipose tissue inflammatory markers. SAA deficiency did not affect lipoprotein cholesterol distributions or plasma triglyceride concentrations in either male or female mice. Atherosclerotic lesion areas measured on the intimal surfaces of the arch, thoracic, and abdominal regions were not significantly different between apoE-/- mice deficient in SAA1.1 and SAA2.1 and apoE-/- mice expressing SAA1.1 and SAA2.1 in either sex. To accelerate lesion formation, mice were fed a Western diet for 12 weeks. SAA deficiency had effect neither on diet-induced alterations in plasma cholesterol, triglyceride, or cytokine concentrations nor on aortic atherosclerotic lesion areas in either male or female mice. In addition, SAA deficiency in male mice had no effect on lesion areas or macrophage accumulation in the aortic roots. CONCLUSIONS: The absence of endogenous SAA1.1 and 2.1 does not affect atherosclerotic lipid deposition in apolipoprotein E-deficient mice fed either normal or Western diets.

Group V secretory phospholipase A2 enhances the progression of angiotensin II-induced abdominal aortic aneurysms but confers protection against angiotensin II-induced cardiac fibrosis in apoE-deficient mice.

Abdominal aortic aneurysms (AAAs) and heart failure are complex life-threatening diseases whose etiology is not completely understood. In this study, we investigated whether deficiency of group V secretory phospholipase A(2) (GV sPLA(2)) protects from experimental AAA. The impact of GV sPLA(2) deficiency on angiotensin (Ang) II-induced cardiac fibrosis was also investigated. Apolipoprotein E (apoE)(-/-) mice and apoE(-/-) mice lacking GV sPLA(2) (GV DKO) were infused with 1000 ng/kg per minute Ang II for up to 28 days. Increases in systolic blood pressure, plasma aldosterone level, and urinary and heart prostanoids were similar in apoE(-/-) and GV DKO mice after Ang II infusion. The incidence of aortic rupture in Ang II-infused GV DKO mice (10%) was significantly reduced compared with apoE(-/-) mice (29.4%). Although the incidence of AAA in GV DKO mice (81.3%) and apoE(-/-) mice (100%) was similar, the mean percentage increase in maximal luminal diameter of abdominal aortas was significantly smaller in GV DKO mice (68.5% ± 7.7%) compared with apoE(-/-) mice (92.6% ± 8.3%). Deficiency of GV sPLA(2) resulted in increased Ang II-induced cardiac fibrosis that was most pronounced in perivascular regions. Perivascular collagen, visualized by picrosirius red staining, was associated with increased TUNEL staining and increased immunopositivity for macrophages and myofibroblasts and nicotinamide adenine dinucleotide phosphate oxidase (NOX)-2 and NOX-4, respectively. Our findings indicate that GV sPLA(2) modulates pathological responses to Ang II, with different outcomes for AAA and cardiac fibrosis.

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages.

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.

Group X secretory phospholipase A2 negatively regulates ABCA1 and ABCG1 expression and cholesterol efflux in macrophages.

OBJECTIVE: GX sPLA(2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids; it has been implicated in inflammatory diseases, including atherosclerosis. To identify a novel role for group X (GX) secretory phospholipase A(2) (sPLA(2)) in modulating ATP binding casette transporter A1 (ABCA1) and ATP binding casette transporter G1 (ABCG1) expression and, therefore, macrophage cholesterol efflux. METHODS AND RESULTS: The overexpression or exogenous addition of GX sPLA(2) significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA(2) deficiency in mouse peritoneal macrophages was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA(2)-overexpressing J774 cells and increased efflux in GX sPLA(2)-deficient mouse peritoneal macrophages. Gene regulation was dependent on GX sPLA(2) catalytic activity, mimicked by arachidonic acid and abrogated when liver X receptor (LXR)α/ß expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA(2) suppresses the ability of LXR to transactivate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand-binding domain. CONCLUSIONS: GX sPLA(2) modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA(2) may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating the genes critical for cellular cholesterol efflux.

Group X secretory phospholipase A2 regulates the expression of steroidogenic acute regulatory protein (StAR) in mouse adrenal glands.

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

Bioactive products generated by group V sPLA(2) hydrolysis of LDL activate macrophages to secrete pro-inflammatory cytokines.

OBJECTIVE: Previous studies have established that hydrolysis of LDL by Group V secretory phospholipase A(2) (GV sPLA(2)) generates a modified particle capable of inducing macrophage foam cell formation. The aim of the present study was to determine whether GV sPLA(2)-hydrolyzed LDL (GV-LDL) produces pro-atherogenic effects in macrophages independent of cholesterol accumulation. METHODS AND RESULTS: J-774 cells incubated with GV-LDL produced more TNF-alpha and IL-6 compared to cells incubated with control-LDL. Indirect immunofluorescence showed that GV-LDL but not control-LDL induced nuclear translocation of NFkappaB. Inhibitors of NFkappaB activation, effectively blocked cytokine production induced by GV-LDL. Control-LDL and GV-LDL were separated from albumin present in reaction mixtures by ultracentrifugation. The albumin fraction derived from GV-LDL contained 80% of the FFA generated and was more potent than the re-isolated GV-LDL in inducing pro-inflammatory cytokine secretion. Linoleic acid (18:2) and oleic acid (18:1) were the most abundant FFAs generated, whereas newly formed lyso-PCs contained 14:0 (myristic), 16:1 (palmitic), and 18:2 fatty acyl groups. Experiments with synthetic FFA showed that 18:1 induced J-774 cells to secrete TNF-alpha and IL-6. CONCLUSIONS: These results indicate that in addition to promoting atherosclerotic lipid accumulation in macrophages, GV sPLA(2) hydrolysis of LDL leads to activation of NFkappaB, a key regulator of inflammation.

Identification and characterization of a cDNA encoding a long-chain cis-isoprenyltranferase involved in dolichyl monophosphate biosynthesis in the ER of brain cells.

A long-chain cis-isoprenyltransferase (cis-IPTase) located in the endoplasmic reticulum (ER) catalyzes the chain elongation stage in the pathway for the de novo biosynthesis of dolichyl monophosphate (Dol-P) in eukaryotic cells. In Saccharomyces cerevisiae, the ER-associated cis-IPTase is encoded by the RER2 gene. Mutations in the RER2 gene result in defects in growth and protein N-glycosylation. In this study a cDNA isolated from human brain (Accession No. AK023164.1), which has substantial homology to cis-IPTases from bacteria, Arabidopsis, and S. cerevisiae, has been shown to: (1) complement the growth defect; (2) restore cis-IPTase activity; dolichol and Dol-P synthesis; and (3) restore normal N-glycosylation of carboxypeptidase Y (CPY) in the yeast rer2Delta mutant. Consistent with a role in Dol-P biosynthesis, overexpression of the human cis-isoprenyltransferase (hCIT) cDNA also suppresses the temperature-sensitive growth and CPY hypoglycosylation phenotypes in sec59-1 cells which are defective in Dol-P biosynthesis due to a temperature-sensitive mutation in dolichol kinase. Overexpression of hCIT in Chinese hamster ovary (CHO) cells results in a modest increase in cis-IPTase activity associated with microsomal fractions and the appearance of a new 38kDa polypeptide that co-localizes with calnexin in the ER, the site of Dol-P biosynthesis, even though no transmembrane domains are predicted by a hydropathy plot.

Expression and characterization of a human cDNA that complements the temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP-mediated kinase activities in Saccharomyces cerevisiae.

Dolichol kinase (DK) catalyzes the CTP-mediated phosphorylation of dolichol in eukaryotic cells, the terminal step in dolichyl monophosphate (Dol-P) biosynthesis de novo. In S. cerevisiae, the SEC59 gene encodes a protein essential for the expression of DK, an enzyme activity that is required for cell viability and normal rates of lipid intermediate synthesis and protein N-glycosylation. This study identifies a cDNA clone from human brain that encodes the mammalian homolog of DK (hDK1p). hDK1 is capable of complementing the growth defect, elevating DK activity, and consequently increasing Dol-P levels in vivo and restoring normal N-glycosylation of carboxypeptidase Y at the restrictive temperature in the temperature-sensitive mutant sec59-1. The CTP-mediated phosphorylation of diacylglycerol (DAG) is unaffected by either the temperature-sensitive mutation in the sec59-1 strain, overexpression of the SEC59 gene, or the mammalian homolog hDK1 under conditions that produced a loss or elevation in the level of DK activity. Additionally, overexpression of hDK1p in Sf-9 cells resulted in a 15-fold increase in DK activity but not DAG kinase activity in crude microsomal fractions. The cloned cDNA contains an open reading frame that would encode a protein with 538 amino acids and a molecular weight of 59,268 kDa. Consistent with this prediction, new polypeptides were detected with an apparent molecular weight of 59-60 kDa when His(6)-tagged constructs of hDK1 or the SEC59 gene were expressed in Sf-9 cells or the temperature-sensitive sec59-1 mutant cells, respectively. These results identify the first cDNA clone encoding a protein required for the expression of DK activity, possibly the catalytic subunit, in a mammalian cell, and establish that the phosphorylation of dolichol and DAG are catalyzed by separate kinase activities in yeast.