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.

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.

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.

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.

Serum amyloid A mediates the inhibitory effect of Ganoderma lucidum polysaccharides on tumor cell adhesion to endothelial cells.

Ganoderma ludicum polysaccharides (GlPS) are the major bioactive composition of Ganoderma lucidum, a well-recognized oriental medical fungus. The published data have shown a complementary effect of GlPS in cancer therapy. The present study was designed to determine the anti-tumor efficacy of GlPS and the possible mechanism covering this effect. Murine Sarcoma 180 (S180) model was established, and GlPS administered orally for 10 days. On the 10th day, tumors were weighed to assess the inhibitory effect of GlPS and sera were collected for proteomic analysis and in vitro study. The in vivo results demonstrated that 25, 50 and 100 mg/kg GlPS inhibited S180 growth by 32.67, 44.80 and 45.24%, respectively (P<0.01). Proteomic study revealed marked protein changes after the process of treatment. Three significantly changed proteins were identified by ESI-Q-TOF-MS and database search indicated that they were haptoblobin, apolipoprotein A-II and serum amyloid A (SAA), respectively. Additionally, the expression change of SAA was confirmed by both Western blot and RT-PCR. The adhesion assay showed that GlPS-treated sera dramatically inhibited the adhesion ability of human prostate carcinoma (PC-3M) cells to human umbilical cord vascular endothelial cells (HUVECs), and this effect partially recovered after immunodepletion by the antibody against SAA. Collectively, these results suggest that GlPS inhibited the tumor growth and tumor cell adhesion to HUVECs via up-regulation of SAA protein expression.

Lovastatin inhibits formation of AA amyloid.

Amyloid A (AA) amyloidosis is a severe complication of many chronic inflammatory disorders, including the hereditary periodic fever syndromes. However, in one of these periodic fever syndromes, the hyper IgD and periodic fever syndrome, amyloidosis is rare despite vigorous, recurring inflammation. This hereditary syndrome is caused by mutations in the gene coding for mevalonate kinase, an enzyme of the isoprenoid pathway. In this study, we used a cell culture system with human monocytes to show that inhibition of the isoprenoid pathway inhibits amyloidogenesis. Inhibition of the isoprenoid pathway by lovastatin resulted in a dose-dependent reduction of amyloid formed [53% at 10 microM (P=0.01)] compared with mononuclear cells that are exposed only to serum AA. The inhibitory effects of lovastatin are reversible by addition of farnesol but not geranylgeraniol. Farnesyl transferase inhibition also inhibited amyloidogenesis. These results implicate that the isoprenoid metabolism could be a potential target for prevention and treatment of AA amyloidosis.

Serum amyloid A-luciferase transgenic mice: response to sepsis, acute arthritis, and contact hypersensitivity and the effects of proteasome inhibition.

Acute phase serum amyloid A proteins (A-SAAs) are multifunctional apolipoproteins produced in large amounts during the acute phase of an inflammation and also during the development of chronic inflammatory diseases. In this study we present a Saa1-luc transgenic mouse model in which SAA1 gene expression can be monitored by measuring luciferase activity using a noninvasive imaging system. When challenged with LPS, TNF-alpha, or IL-1beta, in vivo imaging of Saa1-luc mice showed a 1000- to 3000-fold induction of luciferase activity in the hepatic region that peaked 4-7 h after treatment. The induction of liver luciferase expression was consistent with an increase in SAA1 mRNA in the liver and a dramatic elevation of the serum SAA1 concentration. Ex vivo analyses revealed luciferase induction in many tissues, ranging from several-fold (brain) to >5000-fold (liver) after LPS or TNF-alpha treatment. Pretreatment of mice with the proteasome inhibitor bortezomib significantly suppressed LPS-induced SAA1 expression. These results suggested that proteasome inhibition, perhaps through the NF-kappaB signaling pathway, may regulate SAA1 expression. During the development of acute arthritis triggered by intra-articular administration of zymosan, SAA1 expression was induced both locally at the knee joint and systemically in the liver, and the induction was significantly suppressed by bortezomib. Induction of SAA1 expression was also demonstrated during contact hypersensitivity induced by topical application of oxazolone. These results suggest that both local and systemic induction of A-SAA occur during inflammation and may contribute to the pathogenesis of chronic inflammatory diseases associated with amyloid deposition.

Inhibition of amyloid A amyloidogenesis in vivo and in tissue culture by 4-deoxy analogues of peracetylated 2-acetamido-2-deoxy-alpha- and beta-d-glucose: implications for the treatment of various amyloidoses.

Two novel sugars, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha- and beta-D-xylo-hexopyranoses, have been synthesized and their effects on heparan sulfate biosynthesis using primary mouse hepatocytes in tissue culture have been assessed. At concentrations of 0.1 and 1.0 mmol/L a mixture of both anomers significantly inhibited the biosynthesis of heparan sulfate by 60% and 99%, respectively. At 1.0 mmol/L the average molecular weight of the heparan sulfate synthesized is reduced from 77 kd to 40 kd. The biosynthetic inhibition is apparent within 1 hour (the earliest time point examined) of exposure of the hepatocytes to the analogues and appears virtually complete throughout a 24-hour incubation period. Using a radiolabeled version of the beta-anomer we demonstrate that the analogue is incorporated into growing heparan sulfate chains. The nature of the analogue, the quantity of analogue isotope incorporated, and the reduction in the size of the heparan sulfate polysaccharide are consistent with UDP activation and incorporation of the analogue and truncation of the growing heparan sulfate chain. At 0.1 mmol/L, and in the presence of a constant concentration of serum amyloid A (the precursor to AA amyloid), each analogue inhibited amyloid deposition (by 95 to 99%) in a tissue culture model of AA amyloidogenesis. At 6 mg/dose twice daily each analogue inhibited in vivo splenic AA amyloid deposition by 65 to 70% when using a rapid induction model of mouse AA amyloidogenesis. These data indicate that polysaccharides, such as heparan sulfate, play an integral part in the pathogenesis of AA amyloid deposition, and potentially other forms of amyloid. These data support our previous work that demonstrated that agents that mimic aspects of heparan sulfate structure and that interfere with heparan sulfate:amyloid protein binding inhibit AA amyloid deposition. They emphasize that heparan sulfate likely plays a critical role in amyloidogenesis, and compounds that interfere with heparan sulfate biosynthesis may provide leads for the development of anti-amyloid therapeutic agents.

Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate.

The peroxisome proliferator-activated receptor alpha (PPARalpha), which is highly expressed in liver, plays key roles in lipid metabolism and inflammation. Interleukin-6 (IL-6) is the principal inducer of acute phase response (APR) gene expression. In the present study, we demonstrate that chronic treatment with the PPARalpha agonist fenofibrate fully prevents the IL-6-induced APR gene expression in wild-type but not in PPARalpha-deficient mice. PPARalpha prevents the IL-6-induced expression of the positive APR genes fibrinogen-alpha, -beta, -gamma, haptoglobulin, and serum amyloid A and the IL-6-induced suppression of the negative APR gene, major urinary protein. Furthermore, the effect of PPARalpha on the APR gene expression does not simply consist in a delayed systemic response to IL-6 but occurs directly at the transcriptional level. This global suppression of acute phase gene transcription may be explained by two PPARalpha-dependent in vivo effects: 1) PPARalpha activation results in the down-regulation of the IL-6 receptor components gp80 and gp130 in the liver, thereby reducing the phosphorylation and activation of the downstream transcription factors STAT3 and c-Jun that transduce the IL-6 signal; and 2) PPARalpha reduces the basal expression of the transcription factors CCAAT enhancer-binding protein-alpha, -beta, -delta, which are responsible for immediate and maintained transcription of APR genes. A similar global effect of fenofibrate on acute phase protein expression is observed in hyperlipidemic patients chronically treated with fenofibrate, which displayed decreased plasma concentrations of the positive APR proteins fibrinogen, C-reactive protein, serum amyloid A, plasminogen, and alpha2-macroglobulin and increased plasma concentrations of the negative APR albumin, underlining the clinical significance of our findings.

Adhesion of human platelets to serum amyloid A.

Serum amyloid A (SAA) is an acute phase reactant, and its level in the blood is elevated to 1000-fold in response of the body to trauma, infection, inflammation, and neoplasia. SAA was reported to inhibit platelet aggregation and to induce adhesion of leukocytes. This study looked at adhesion of human platelets to SAA. Immobilized SAA supported the adhesion of human washed platelets; level of adhesion to SAA was comparable to fibronectin and lower than to fibrinogen. Adhesion to SAA was further enhanced by Mn(2+) and the physiological agonist, thrombin. Platelet adhesion to SAA was completely abolished by anti-SAA antibody. SAA-induced adhesion was inhibited by antibodies against the integrin receptor alphaIIbbeta3, by the peptide GRGDSP and by SAA-derived peptide containing YIGSR-like and RGD-like adhesion motifs (amino acids 29 to 42). Adhesion was not inhibited by control immunoglobulin G, by antibody against the integrin receptor alphaVbeta3, by the peptide GRGESP, and by SAA-derived peptide that includes incomplete RGD motif. SAA-derived peptide 29 to 42 also inhibited platelet adhesion to fibronectin. Transfected human melanoma cells expressing alphaIIbbeta3 adhered to SAA, whereas transfected cells expressing alphaVbeta3 did not. By using flow cytometry, the alphaIIbbeta3 cells displayed significantly higher levels of binding of soluble SAA than the alphaVbeta3 cells. These data indicate that human platelets specifically adhere to SAA in an RGD- and alphaIIbbeta3-dependent manner. Thus, SAA may play a role in modulating platelet adhesion at vascular injury sites by sharing platelet receptors with other platelet-adhesive proteins.

Effect of poly(vinylsulfonate) on murine AA amyloid: a high-resolution ultrastructural study.

In experimental murine inflammation-associated amyloidosis (AA amyloidosis), an interaction between heparan sulfate and serum amyloid A (SAA), the AA precursor, has been demonstrated and is believed to play an important role in AA amyloidogenesis. Poly(vinylsulfonate) sodium salt (PVS) can arrest AA amyloid induction and cause established amyloid deposits to regress. PVS is thought to have this property by virtue of limited anionic structural similarities it has to heparan sulfate. In the present study, a comparison has been made of the in situ light microscopic and high-resolution ultrastructure of amyloid deposits before and after PVS treatment. As shown recently in situ, AA fibrils from untreated mice are composed of an outer layer of heparan sulfate proteoglycan and a 1- to 2-nm filament network of AA protein. This layer encloses a microfibril-like structure composed of chondroitin sulfate proteoglycan wound around a core of amyloid P component. After treatment with PVS, both the heparan sulfate proteoglycan and the AA filament network are lost from the fibrils, and the more central portion disintegrates into the chondroitin sulfate proteoglycan with associated amyloid P subunits. These findings add further support to the concept that heparan sulfate proteoglycan is important in amyloid fibril structure, and interference with its binding interactions with the amyloid filament protein provides a point of therapeutic attack.