Serum amyloid A1: Innocent bystander or active participant in cell migration in triple-negative breast cancer?

The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G2/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1ß, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

Inhibition of IL-6 in the LCWE Mouse Model of Kawasaki Disease Inhibits Acute Phase Reactant Serum Amyloid A but Fails to Attenuate Vasculitis.

Objective: Kawasaki disease (KD) is the most common cause of acquired pediatric heart disease in the developed world. 10% of KD patients are resistant to front-line therapy, and no interventions exist to address secondary complications such as myocardial fibrosis. We sought to identify proteins and pathways associated with disease and anti-IL-1 treatment in a mouse model of KD. Methods: Vasculitis was induced via Lactobacillus casei cell wall extract (LCWE) injection in 5-week-old male mice. Groups of mice were injected with LCWE alone, LCWE and IL-1 receptor antagonist anakinra, or saline for controls. Upper heart tissue was assessed by quantitative mass spectrometry analysis. Expression and activation of STAT3 was assessed by immunohistochemistry, immunofluorescence and Western blot, and IL-6 expression by RNA-seq and ELISA. A STAT3 small molecular inhibitor and anti-IL-6R antibody were used to evaluate the role of STAT3 and IL-6 in disease development. Results: STAT3 was highly expressed and phosphorylated in cardiac tissue of LCWE-injected mice, and reduced following anakinra treatment. Il6 and Stat3 gene expression was enhanced in abdominal aorta of LCWE-injected mice and reduced with Anakinra treatment. IL-6 serum levels were enhanced in LCWE-injected mice and normalized by anakinra. However, neither inhibition of STAT3 nor blockade of IL-6 altered disease development. Conclusion: Proteomic analysis of cardiac tissues demonstrates differential protein expression between KD-like, control and anakinra treated cardiac tissue. STAT3 and IL-6 were highly upregulated with LCWE and normalized by anakinra treatment. However, both STAT3 and IL-6 were dispensable for disease development indicating they may be bystanders of inflammation.

Cell assay for the identification of amyloid inhibitors in systemic AA amyloidosis.

Systemic AA amyloidosis is still, up to this day, a life-threatening complication of chronic inflammatory diseases. Despite the success of anti-inflammatory treatment, the prognosis of some AA patients is still poor, which is why therapies directed at the amyloidogenic pathway in AA amyloidosis are being sought after. The cell culture model of amyloid formation from serum amyloid A1 (SAA1) protein remodels crucial features of AA amyloid deposit formation in vivo. We here demonstrate how the cell model can be utilized for the identification of compounds with amyloid inhibitory activity. Out of five compounds previously reported to inhibit self-assembly of various amyloidogenic proteins, we found that epigallocatechin gallate (EGCG) inhibited the formation of SAA1-derived fibrils in cell culture. From a series of compounds targeting the protein quality control machinery, the autophagy inhibitor wortmannin reduced amyloid formation, while the other tested compounds did not lead to a substantial reduction of the amyloid load. These data suggest that amyloid formation can be targeted not only via the protein self-assembly pathway directly, but also by treatment with compounds that impact the cellular protein machinery.

Nanotherapy for Alzheimer's disease and vascular dementia: Targeting senile endothelium.

Due to the complexity of Alzheimer's disease, multiple cellular types need to be targeted simultaneously in order for a given therapy to demonstrate any major effectiveness. Ultrasound-sensitive coated microbubbles (in a targeted lipid nanoemulsion) are available. Versatile small molecule drug(s) targeting multiple pathways of Alzheimer's disease pathogenesis are known. By incorporating such drug(s) into the targeted "lipid-coated microbubble" [LCM]/"nanoparticle-derived" [ND] (or LCM/ND) nanoemulsion type, one obtains a multitasking combination therapeutic for translational medicine. This multitasking therapeutic targets cell-surface scavenger receptors (mainly class B type I), or SR-BI, making possible for various Alzheimer's-related cell types to be simultaneously searched out for localized drug treatment in vivo. Besides targeting cell-surface SR-BI, the proposed LCM/ND-nanoemulsion combination therapeutic(s) include a characteristic lipid-coated microbubble [LCM] subpopulation (i.e., a stable LCM suspension); such film-stabilized microbubbles are well known to substantially reduce the acoustic power levels needed for accomplishing temporary noninvasive (transcranial) ultrasound treatment, or sonoporation, if additionally desired for the Alzheimer's patient.

Effect of statins on atherogenic serum amyloid A and α1-antitrypsin low-density lipoprotein complexes.

PURPOSE: HMG-CoA reductase inhibitors, also termed statins, are used to reduce the risk of coronary artery disease. Two oxidatively modified low-density lipoprotein (LDL) complexes, serum amyloid A-LDL (SAA-LDL) and α1-antitrypsin-LDL (AT-LDL), serve as atherosclerotic, inflammatory, and cardiovascular risk markers. In this study, we examined the effects of hydrophilic rosuvastatin (RSV) and lipophilic pitavastatin (PTV) on these markers in patients with hypercholesterolemia. METHODS: The present study was a sub-analysis of our previous STAT-LVDF study. The subjects were treated with RSV or PTV for 24weeks. Changes in glucose-lipid metabolism, serum levels of SAA-LDL and AT-LDL, and C-reactive protein (CRP) level were assessed. RESULTS: In total, 53 patients were analyzed in the present study. RSV and PTV significantly decreased SAA-LDL (RSV: p=0.003, PTV: p=0.012) and AT-LDL levels (RSV: p=0.013, PTV: p=0.037). Changes in SAA-LDL level were significantly and positively correlated with those in CRP in both the RSV (r=0.549, p=0.003) and PTV (r=0.576, p=0.004) groups. Moreover, a positive correlation between changes of SAA-LDL levels and those of HbA1c levels was observed in the PTV group (r=0.442, p=0.030) but not in the RSV group (r=-0.100, p=0.611). CONCLUSIONS: Both hydrophilic rosuvastatin and lipophilic pitavastatin reduce serum levels of atherosclerotic and inflammatory markers. These findings also indicate differential effects of RSV and PTV on glucose tolerance.

Designing peptidic inhibitors of serum amyloid A aggregation process.

Amyloid A amyloidosis is a life-threatening complication of a wide range of chronic inflammatory, infectious and neoplastic diseases, and the most common form of systemic amyloidosis worldwide. It is characterized by extracellular tissue deposition of fibrils that are composed of fragments of serum amyloid A protein (SAA), a major acute-phase reactant protein, produced predominantly by hepatocytes. Currently, there are no approved therapeutic agents directed against the formation of fibrillar SAA assemblies. We attempted to develop peptidic inhibitors based on their similarity and complementarity to the regions critical for SAA self-association, which they should interact with and block their assembly into amyloid fibrils. Inh1 and inh4 which are comprised of the residues from the amyloidogenic region of SAA1.1 protein and Aß peptide, respectively, were found by us as capable to significantly suppress aggregation of the SAA1-12 peptide. It was chosen as an aggregation model that mimicks the amyloidogenic nucleus of SAA protein. We suppose that aromatic interactions may be responsible for inhibitory activity of both compounds. We also recognized that aromatic residues are involved in self-association of SAA1-12.

Serum amyloid A receptor blockade and incorporation into high-density lipoprotein modulates its pro-inflammatory and pro-thrombotic activities on vascular endothelial cells.

The acute phase protein serum amyloid A (SAA), a marker of inflammation, induces expression of pro-inflammatory and pro-thrombotic mediators including ICAM-1, VCAM-1, IL-6, IL-8, MCP-1 and tissue factor (TF) in both monocytes/macrophages and endothelial cells, and induces endothelial dysfunction-a precursor to atherosclerosis. In this study, we determined the effect of pharmacological inhibition of known SAA receptors on pro-inflammatory and pro-thrombotic activities of SAA in human carotid artery endothelial cells (HCtAEC). HCtAEC were pre-treated with inhibitors of formyl peptide receptor-like-1 (FPRL-1), WRW4; receptor for advanced glycation-endproducts (RAGE), (endogenous secretory RAGE; esRAGE) and toll-like receptors-2/4 (TLR2/4) (OxPapC), before stimulation by added SAA. Inhibitor activity was also compared to high-density lipoprotein (HDL), a known inhibitor of SAA-induced effects on endothelial cells. SAA significantly increased gene expression of TF, NFκB and TNF and protein levels of TF and VEGF in HCtAEC. These effects were inhibited to variable extents by WRW4, esRAGE and OxPapC either alone or in combination, suggesting involvement of endothelial cell SAA receptors in pro-atherogenic gene expression. In contrast, HDL consistently showed the greatest inhibitory action, and often abrogated SAA-mediated responses. Increasing HDL levels relative to circulating free SAA may prevent SAA-mediated endothelial dysfunction and ameliorate atherogenesis.

A potential role for regulatory T-cells in the amelioration of DSS induced colitis by dietary non-digestible polysaccharides.

Inflammatory bowel diseases (IBD) including ulcerative colitis (UC) and Crohn's disease (CD) are chronic relapsing inflammatory disorders of the gastrointestinal tract. The interaction between a disturbed microbial composition, the intestinal mucosal barrier and the mucosal immune system plays an important role in IBD and its chronicity. It has been indicated that due to the altered microbial composition the balance between T regulatory cells (Treg) and T helper cells (Th) 17 is disturbed, leading to an inflammatory state. The present study shows that oral intake of a specific multi fibre mix (MF), designed to match the fibre content of a healthy diet, counteracts IBD-like intestinal inflammation and weight loss in dextran sodium sulphate treated mice. This reduction in inflammation might be brought about, at least in part, by the MF-induced decrease in inflammatory cytokines, increase in IL-10 and the relative increase in Treg cells in the mesenteric lymph nodes (MLN). Moreover, the Treg percentage in the MLN correlates with the percentage of tolerogenic lamina propria derived CD103+RALDH+dendritic cells in the MLN, suggesting that these play a role in the observed effects. In children with CD exclusive enteral nutrition (EEN) is a widely used safe and effective therapy. Optimizing enteral nutritional concepts with the tested fibre mix, know to modulate the gut microbiota composition, SCFA production and inflammatory status (as indicated by the present study) could possibly further improve efficacy in inducing remission.

Acute-phase protein serum amyloid A3 is a novel paracrine coupling factor that controls bone homeostasis.

Serum amyloid A (A-SAA/Saa3) was shown before to affect osteoblastic metabolism. Here, using RT-quantitative PCR and/or immunoblotting, we show that expression of mouse Saa3 and human SAA1 and SAA2 positively correlates with increased cellular maturation toward the osteocyte phenotype. Expression is not detected in C3H10T1/2 embryonic fibroblasts but is successively higher in preosteoblastic MC3T3-E1 cells, late osteoblastic MLO-A5 cells, and MLO-Y4 osteocytes, consistent with findings using primary bone cells from newborn mouse calvaria. Recombinant Saa3 protein functionally inhibits osteoblast differentiation as reflected by reductions in the expression of osteoblast markers and decreased mineralization in newborn mouse calvaria. Yet, Saa3 protein enhances osteoclastogenesis in mouse macrophages/monocytes based on the number of multinucleated and tartrate-resistant alkaline phosphatase-positive cells and Calcr mRNA expression. Depletion of Saa3 in MLO osteocytes results in the loss of the mature osteocyte phenotype. Recombinant osteocalcin, which is reciprocally regulated with Saa3 at the osteoblast/osteocyte transition, attenuates Saa3 expression in MLO-Y4 osteocytes. Mechanistically, Saa3 produced by MLO-Y4 osteocytes is integrated into the extracellular matrix of MC3T3-E1 osteoblasts, where it associates with the P2 purinergic receptor P2rx7 to stimulate Mmp13 expression via the P2rx7/MAPK/ERK/activator protein 1 axis. Our data suggest that Saa3 may function as an important coupling factor in bone development and homeostasis.

An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo.

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

S100A12 suppresses pro-inflammatory, but not pro-thrombotic functions of serum amyloid A.

S100A12 is elevated in the circulation in patients with chronic inflammatory diseases and recent studies indicate pleiotropic functions. Serum amyloid A induces monocyte cytokines and tissue factor. S100A12 did not stimulate IL-6, IL-8, IL-1ß or TNF-α production by human peripheral blood mononuclear cells but low amounts consistently reduced cytokine mRNA and protein levels induced by serum amyloid A, by ∼49% and ∼46%, respectively. However, S100A12 did not affect serum amyloid A-induced monocyte tissue factor. In marked contrast, LPS-induced cytokines or tissue factor were not suppressed by S100A12. S100A12 did not alter cytokine mRNA stability or the cytokine secretory pathway. S100A12 and serum amyloid A did not appear to form complexes and although they may have common receptors, suppression was unlikely via receptor competition. Serum amyloid A induces cytokines via activation of NF-κB and the MAPK pathways. S100A12 reduced serum amyloid A-, but not LPS-induced ERK1/2 phosphorylation to baseline. It did not affect JNK or p38 phosphorylation or the NF-κB pathway. Reduction in ERK1/2 phosphorylation by S100A12 was unlikely due to changes in intracellular reactive oxygen species, Ca(2+) flux or to recruitment of phosphatases. We suggest that S100A12 may modulate sterile inflammation by blunting pro-inflammatory properties of lipid-poor serum amyloid A deposited in chronic lesions where both proteins are elevated as a consequence of macrophage activation.

Homocysteine induces serum amyloid A3 in osteoblasts via unlocking RGD-motifs in collagen.

Hyperhomocysteinemia is a risk factor for osteoporotic fractures. Homocysteine (Hcys) inhibits collagen cross-linking and consequently decreases bone extracellular matrix (ECM) quality. Serum amyloid A (A-SAA), an acute-phase protein family, plays an important role in chronic and inflammatory diseases and up-regulates MMP13, which plays an important role in bone development and remodeling. Here, we investigate the effect of Hcys on expression of SAA3, a member of the A-SAA gene family, in osteoblasts characterizing underlying mechanisms and possible consequences on bone metabolism. MC3T3-E1 osteoblast-like cells were cultured up to 21 d with Hcys (low millimolar range) or reseeded onto ECM resulting from untreated or Hcys-treated MC3T3-E1 cells. Fourier-transformed infrared spectroscopy and a discriminative antibody were used to characterize the resulting ECM. Gene expression and signaling pathways were analyzed by gene chip, quantitative RT-PCR, and immunoblotting. Transcriptional regulation of Saa3 was studied by promoter transfection assays, chromatin immunoprecipitation, and immunofluorescence microscopy. Hcys treatment resulted in reduced collagen cross-linking, uncovering of RGD-motifs, and activation of the PTK2-PXN-CTNNB1 pathway followed by RELA activation. These signaling events led to increased SAA3 expression followed by the production of MMP13 and several chemokines, including Ccl5, Ccl2, Cxcl10, and Il6. Our data suggest Saa3 as link between hyperhomocysteinemia and development of osteoporosis.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells.

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

Aberrant expression of serum amyloid A in head and neck squamous cell carcinoma.

Serum amyloid A (SAA) is an acute-phase reactant, the blood level of which is often elevated in response to some types of neoplasia. Here, we investigated expression of the gene SAA1 and the protein SAA in head and neck squamous cell carcinoma (HNSCC) and normal oral mucosal tissues as well as blood SAA levels in HNSCC patients. Also, we investigated the effects of inhibiting signal transducer and activator of transcription 3 (STAT3) signaling on SAA1 expression in the HNSCC cell line SAS. Serum SAA levels in HNSCC patients were significantly higher than those in healthy volunteers. In addition, real-time quantitative reverse transcription-polymerase chain reaction analysis revealed a significantly higher SAA1 expression level in HNSCC than in normal mucosa (P < 0.0001). Furthermore, Western blot and immunohistochemical analyzes revealed that high expression of SAA in carcinomas was detected predominantly in tumor cells, but not in normal mucosal tissues. An inhibitor of STAT3 activation, AG490, significantly reduced SAA1 expression in SAS cells. These data demonstrated that SAA was up-regulated in HNSCC through the Janus kinase-STAT3 pathway.

LL-37 inhibits serum amyloid A-induced IL-8 production in human neutrophils.

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.

Continuous positive nasal airway pressure decreases levels of serum amyloid A and improves autonomic function in obstructive sleep apnea syndrome.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is associated with the pathogenesis of cardiovascular disease, and inflammation and autonomic dysfunction. We investigated levels of serum amyloid A (SAA), a marker of inflammation, as well as autonomic nervous activity and pulse wave velocity (PWV) before and after nasal continuous positive airway pressure (nCPAP) therapy in patients with obstructive sleep apnea. METHODS AND RESULTS: We separated 116 patients who were diagnosed with OSAS by polysomnography according to the apnea hypopnea index (AHI) into the following groups: Group 1 without or with mild OSAS (AHI<20, n=35), Group 2 with moderate OSAS (20==40, n=46). Serum level of SAA (p<0.05), brachial-ankle PWV (p<0.05) and BP (p<0.005) were significantly higher in Group 3 than in Group 1. Autonomic nervous activity assessed by autoregressive spectral analysis of heart rate variability showed that high frequency (HF) power, an indicator of vagal activity, was decreased in Groups 2 and 3 (p<0.05) and that low frequency/HF, an indicator of sympathetic activity, was increased in Group 3 (p<0.05). After 3 months of nCPAP therapy in Group 3 (n=38), SAA (p<0.05), PWV (p<0.001) and BP (p<0.05) were significantly decreased. CONCLUSION: Markers of inflammation and autonomic dysfunction are increased in patients with OSAS, and nCPAP might help to reduce these risk factors for cardiovascular diseases.

LL-37 inhibits serum amyloid A-induced IL-8 production in human neutrophils

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.