Pathogenetic mechanisms of amyloid A amyloidosis

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.     

Acceleration of murine AA amyloid deposition by bovine amyloid fibrils and tissue homogenates

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported for several types of amyloidosis. Reactive amyloidosis (AA) occurs in a wide variety of domestic animal species and is characterized by amyloid deposition mainly in spleen, liver, and kidneys. Because the visceral organs of domestic animals have traditionally been used in Asian cuisines, it is important to examine whether dietary ingestion of the organs themselves (rather than purified amyloid fibrils) accelerates AA amyloid deposition. Herein, we show that murine AA amyloidosis develops rapidly after intraperitoneal or oral administration of purified amyloid fibrils or homogenates of amyloid-laden bovine liver. The amyloidosis development in mice was dependent on the concentration of amyloid fibrils or amyloidotic liver homogenates. We found that experimental murine AA amyloidosis was accelerated by dietary ingestion of both purified amyloid fibrils and tissue homogenates that contain amyloid fibrils. We also investigated livers of beef cattle and food chickens to examine whether they contain amyloid-enhancing factor activity. By microscopic examination of hematoxylin and eosin- and Congo red-stained sections, no amyloid deposition was detected in these livers, and no effective activity for experimental induction of AA amyloidosis in mice was detected in homogenates of these livers.     

Heparanase, tissue factor, and cancer

Heparanase is an endo-beta- D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Evidence was provided that heparanase overexpression in human leukemia, glioma, and breast carcinoma cells results in a marked increase in tissue factor (TF) levels. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. TF induction was further confirmed in heparanase-overexpressing transgenic mice and correlated with heparanase expression levels in leukemia patients. Heparanase was also found to be involved in the regulation of tissue factor pathway inhibitor (TFPI). It was shown that heparanase overexpression or exogenous addition induces two- to threefold increase of TFPI expression. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. Extracellular accumulation exceeded, however, the observed increase in TFPI at the protein level and appeared to be independent of heparan sulfate and heparanase enzymatic activity. Instead, a physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local prothrombotic function of heparanase. As heparins are strong inhibitors of heparanase, in view of the effect of heparanase on TF/TFPI pathway, the role of heparins' anticoagulant activity may potentially be expanded.     

Slaughtered aged cattle might be one dietary source exhibiting amyloid enhancing factor activity

It has been shown that experimental murine AA amyloidosis can be enhanced by dietary ingestion of amyloid fibrils, and it is known that systemic AA amyloidosis occasionally develops in aged cattle. In this study, we examined amyloid deposits in renal and muscular tissues simultaneously obtained from slaughtered aged cattle; from both tissues when affected, amyloid-enhancing activity was also investigated. On histopathology, renal amyloid deposition was seen in nine of the 293 cattle with no history of disease, and minute amyloid deposition in muscular tissue was detectable in one of these nine. All these amyloid deposits were immunohistochemically demonstrated to be AA. Extracts, which might contain amyloid fibril fractions, were isolated from renal and muscular tissues in five of these nine cattle. On SDS-PAGE and Western blot analysis, protein bands immunoreactive to anti-AA serum were detected in the kidney fractions obtained from four of the five latter cattle, but no bands were seen in the muscle fractions of any of the five cattle. Amyloid fibril fractions from two cattle were intravenously injected into group of seven experimentally designed mice for induction of AA amyloidosis. All seven mice injected with kidney fraction developed severe AA amyloidosis, whereas only one of the seven mice given muscle fraction showed slight amyloid deposition in the spleen. These data suggest that food products made from aged cattle possess amyloid-enhancing potential.     

Formation of experimental murine AA amyloid fibrils in SAP-deficient mice: High resolution ultrastructural study

Previously, the role of the serum amyloid P component (SAP) in the deposition of murine AA amyloid has been examined in SAP-deficient mice in which the deposition was significantly retarded. In this study, AA amyloid fibrillogenesis in SAP-deficient mice was examined ultrastructurally. The fibrils of wild type mice were made up of a microfibril-like main body composed of SAP, chondroitin sulfate proteoglycan (CSPG), and outermost heparan sulfate proteoglycan (HSPG), and associated on its surface were 3 nm wide AA protein ‘helical rods’, a possible suitable form for Congo red staining. In SAP-deficient mice, fibrils of a similar appearance were also noted among an overwhelming amount of amorphous material, but the AP-containing main body of the fibril was replaced by elongated irregular aggregates of CSPG. The mechanism of retardation of AA amyloid induction in SAP-deficient mice has not yet been clear. It may be caused by possible slower formation of a ‘substitute’ core. Also, slower formation of AA helical rods may be possible due to the difference in the core material to which AA protein is attached. If it is so, it may limit the extent of Congo red staining, resulting in underestimation of the actual amount of AA protein.     

Formation of experimental murine AA amyloid fibrils in SAP-deficient mice: high resolution ultrastructural study.

Previously, the role of the serum amyloid P component (SAP) in the deposition of murine AA amyloid has been examined in SAP-deficient mice in which the deposition was significantly retarded. In this study, AA amyloid fibrillogenesis in SAP-deficient mice was examined ultrastructurally. The fibrils of wild type mice were made up of a microfibril-like main body composed of SAP, chondroitin sulfate proteoglycan (CSPG), and outermost heparan sulfate proteoglycan (HSPG), and associated on its surface were 3 nm wide AA protein 'helical rods', a possible suitable form for Congo red staining. In SAP-deficient mice, fibrils of a similar appearance were also noted among an overwhelming amount of amorphous material, but the AP-containing main body of the fibril was replaced by elongated irregular aggregates of CSPG. The mechanism of retardation of AA amyloid induction in SAP-deficient mice has not yet been clear. It may be caused by possible slower formation of a 'substitute' core. Also, slower formation of AA helical rods may be possible due to the difference in the core material to which AA protein is attached. If it is so, it may limit the extent of Congo red staining, resulting in underestimation of the actual amount of AA protein.     

Quantitative high-resolution microradiographic imaging of amyloid deposits in a novel murine model of AA amyloidosis

The mouse model of experimentally induced systemic AA amyloidosis is long established, well validated, and closely analogous to the human form of this disease. However, the induction of amyloid by experimental inflammation is unpredictable, inconsistent, and difficult to modulate. We have previously shown that murine AA amyloid deposits can be imaged using iodine-123 labeled SAP scintigraphy and report here substantial refinements in both the imaging technology and the mouse model itself. In this regard, we have generated a novel prototype of AA amyloid in which mice expressing the human interleukin 6 gene, when given amyloid enhancing factor, develop extensive and progressive systemic AA deposition without an inflammatory stimulus, i.e., a transgenic rapidly inducible amyloid disease (TRIAD) mouse. Additionally, we have constructed high-resolution micro single photon emission computed tomography (SPECT)/computed tomography (CT) instrumentation that provides images revealing the precise anatomic location of amyloid deposits labeled by radioiodinated serum amyloid P component (SAP). Based on reconstructed microSPECT/CT images, as well as autoradiographic, isotope biodistribution, and quantitative histochemical analyses, the ¹²⁵I-labeled SAP tracer bound specifically to hepatic and splenic amyloid in the TRIAD animals. The ability to discern radiographically the extent of amyloid burden in the TRIAD model provides a unique opportunity to evaluate the therapeutic efficacy of pharmacologic compounds designed to inhibit fibril formation or effect amyloid resolution.     

FK506 inhibits murine AA amyloidosis: possible involvement of T cells in amyloidogenesis

OBJECTIVE: To determine the possibility that T cells represent a potential target for therapy in AA amyloidosis. METHODS: AA amyloidosis was induced in C3H/HeN mice by concomitant administration of AgNO3 and amyloid-enhancing factor (AEF). Mice injected with AgNO3 and AEF received intraperitoneal injections of FK506 (2-200 microg/day). The degree of splenic amyloid deposition was determined by Congo red staining. Serum amyloid A (SAA), interleukin 1beta (IL-1beta), IL-6, and tumor necrosis factor-a concentrations were measured by ELISA. AA amyloidosis was also induced in ICR mice by injection of Freund's complete adjuvant (FCA) and Mycobacterium butyricum without AEF. ICR mice injected with FCA and M. butyricum also received intraperitoneal injections of FK506 (200 microg/day) to eliminate the possibility that FK506 action might depend upon AEF activity in the amyloid formation. Amyloid deposition was also induced with and without AEF in severe combined immunodeficient (SCID) mice and nude mice to clarify the role of T cells in the mechanism of amyloid formation in AA amyloidosis. RESULTS: FK506 treatment significantly reduced the amount of amyloid deposition and incidence of amyloidosis without reducing serum SAA and proinflammatory cytokine levels in the murine AA amyloidosis models with and without AEF. SCID mice and nude mice showed resistance to development of AA amyloidosis. CONCLUSION: Our findings may provide a new therapeutic strategy for amyloidosis. The results suggested that T cells may play an important role in the mechanism of amyloid formation in AA amyloidosis.     

Quantitative high-resolution microradiographic imaging of amyloid deposits in a novel murine model of AA amyloidosis.

The mouse model of experimentally induced systemic AA amyloidosis is long established, well validated, and closely analogous to the human form of this disease. However, the induction of amyloid by experimental inflammation is unpredictable, inconsistent, and difficult to modulate. We have previously shown that murine AA amyloid deposits can be imaged using iodine-123 labeled SAP scintigraphy and report here substantial refinements in both the imaging technology and the mouse model itself. In this regard, we have generated a novel prototype of AA amyloid in which mice expressing the human interleukin 6 gene, when given amyloid enhancing factor, develop extensive and progressive systemic AA deposition without an inflammatory stimulus, i.e., a transgenic rapidly inducible amyloid disease (TRIAD) mouse. Additionally, we have constructed high-resolution micro single photon emission computed tomography (SPECT)/computed tomography (CT) instrumentation that provides images revealing the precise anatomic location of amyloid deposits labeled by radioiodinated serum amyloid P component (SAP). Based on reconstructed microSPECT/CT images, as well as autoradiographic, isotope biodistribution, and quantitative histochemical analyses, the (125)I-labeled SAP tracer bound specifically to hepatic and splenic amyloid in the TRIAD animals. The ability to discern radiographically the extent of amyloid burden in the TRIAD model provides a unique opportunity to evaluate the therapeutic efficacy of pharmacologic compounds designed to inhibit fibril formation or effect amyloid resolution.     

Homozygous serum amyloid P component-deficiency does not enhance regression of AA amyloid deposits.

Serum amyloid P component (SAP) is a common protein constituent of all types of amyloid deposits. Using SAP-deficient mice generated through gene targeting, we and others have shown that SAP significantly promotes amyloid deposition. It has been speculated that SAP protects amyloid fibrils from degradation by coating their exterior surface. To assess potential ways of treating individuals with amyloidosis, we examined the persistence of splenic AA amyloid fibrils in SAP-deficient and wild-type mice. No enhancement in the rate of regression of splenic AA amyloid was observed in the SAP-deficient mice relative to wild-type mice. These results present, for the first time, evidence that lack of SAP in AA amyloid deposits does not enhance regression of the deposits in vivo and suggest that dissociation of bound SAP from AA amyloid deposits would not significantly accelerate regression of the deposits in vivo.     

Endoscopic and histopathological features of gastrointestinal amyloidosis

Amyloidosis is a rare disorder, characterized by the extracellular deposition of an abnormal fibrillar protein, which disrupts tissue structure and function. Amyloidosis can be acquired or hereditary, and systemic or localized to a single organ, such as the gastrointestinal (GI) tract. Clinical manifestations may vary from asymptomatic to fatal forms. Primary amyloidosis (monoclonal immunoglobulin light chains, AL) is the most common form of amyloidosis. AL amyloidosis has been associated with plasma cell dyscrasias, such as, mul- tiple myeloma. Secondary amyloidosis is caused by the deposition of fragments of the circulating acute-phase reactant, serum amyloid A protein (SAA). Common causes of AA amyloidosis are chronic inflammatory dis-orders. Although GI symptoms are usually nonspecific, histopathological patterns of amyloid deposition are associated with clinical and endoscopic features. Amyloid deposition in the muscularis mucosae, submucosa, and muscularis propria has been dominant in AL amyloidosis, leading to polypoid protrusions and thickening of the valvulae conniventes, whereas granular amyloid deposition mainly in the propria mucosae has been related to AA amyloidosis, resulting in the fine granular appearance, mucosal friability, and erosions. As a result, AL amyloidosis usually presents with constipation, mechanical obstruction, or chronic intestinal pseudoobstruction while AA amyloidosis presents with diarrhea and malabsorption Amyloidotic GI symptoms are mostly refractory and have a negative impact on quality of life and survival. Diagnosing GI amyloidosis requires high suspicion of evaluating endoscopists. Because of the absence of specific treatments for reducing the abundance of the amyloidogenic precursor protein, we should be aware of certain associations between patterns of amyloid deposition and clinical and endoscopic features.     

Acceleration of murine amyloidosis by implantation of amyloid-containing grafts.

We examined the transmissibility of amyloidosis by the implantation of amyloid-containing tissue. If the transmissibility similar to prion diseases is applicable, using amyloid-containing tissue for transplantation in humans might be a risk factor. In this study, AA amyloidosis occurred in mice that underwent implantation of AA amyloid-containing grafts to the liver and subsequent inflammatory stimulation. AApoAII amyloidosis occurred after implantation of AApoAII amyloid-containing grafts to the liver or to the subcutaneous space without inflammatory stimulation. Both types of amyloidoses occurred in the recipient mice sooner than expected. Moreover, AA and AApoAII amyloid deposits were found at 12 weeks after implantation in mice given AApoAII amyloid-containing grafts and inflammatory stimulation. These results suggest that implanted amyloid deposits have an AEF effect and that implanted amyloid-containing tissue can promote and accelerate a different type of amyloidosis. In another experiment, mice received amyloid-containing or normal tissue grafts. The degree of amyloid deposition was compared after 6 days and 5 weeks of inflammatory stimulation and when the mice were killed. There was no obvious difference in the degree of amyloid deposition between each group, indicating that the lag-time is shortened by implantation of amyloid-containing tissue, resulting in severe amyloidosis in the short term.     

Effect of heating on the stability of amyloid A (AA) fibrils and the intra- and cross-species transmission of AA amyloidosis

Amyloid A (AA) amyloidosis is a protein misfolding disease characterized by extracellular deposition of AA fibrils. AA fibrils are found in several tissues from food animals with AA amyloidosis. For hygienic purposes, heating is widely used to inactivate microbes in food, but it is uncertain whether heating is sufficient to inactivate AA fibrils and prevent intra- or cross-species transmission. We examined the effect of heating (at 60?°C or 100?°C) and autoclaving (at 121?°C or 135?°C) on murine and bovine AA fibrils using Western blot analysis, transmission electron microscopy (TEM), and mouse model transmission experiments. TEM revealed that a mixture of AA fibrils and amorphous aggregates appeared after heating at 100?°C, whereas autoclaving at 135?°C produced large amorphous aggregates. AA fibrils retained antigen specificity in Western blot analysis when heated at 100?°C or autoclaved at 121?°C, but not when autoclaved at 135?°C. Transmissible pathogenicity of murine and bovine AA fibrils subjected to heating (at 60?°C or 100?°C) was significantly stimulated and resulted in amyloid deposition in mice. Autoclaving of murine AA fibrils at 121?°C or 135?°C significantly decreased amyloid deposition. Moreover, amyloid deposition in mice injected with murine AA fibrils was more severe than that in mice injected with bovine AA fibrils. Bovine AA fibrils autoclaved at 121?°C or 135?°C did not induce amyloid deposition in mice. These results suggest that AA fibrils are relatively heat stable and that similar to prions, autoclaving at 135?°C is required to destroy the pathogenicity of AA fibrils. These findings may contribute to the prevention of AA fibril transmission through food materials to different animals and especially to humans.     

Rapid induction of experimental AA amyloidosis in mink by intravenous injection of amyloid enhancing factor

Studies of amyloid enhancing factor (AEF)-induced amyloidosis are commonly performed in mice. In mink, earlier studies of amyloid A (AA) amyloidosis showed that the predeposition phase was highly variable. Thus, the aim of the study was to establish an AEF-induced AA amyloidosis model in mink to facilitate studies of early amyloid deposition in a species with prominent ellipsoids, anatomical structures lacking in mice but present in most other mammals. AEF was extracted from mink spleens containing AA. Mink received one intravenous injection of AEF and repeated subcutaneous injections of lipopolysaccharide (LPS) as an inflammatory stimulus. On day 4, small amounts of amyloid were detected in the marginal zone in the spleen. On day 7, considerable amyloid deposition was detected in the ellipsoids and marginal zones in the spleen and in the space of Disse in the liver. By immunohistochemistry, the deposits were identified as AA amyloid. Immunolabeling was also detected in lymphoid follicles and the red pulp of some animals. Control animals receiving only AEF were negative. Control animals receiving only LPS were negative except for one of three animals which had small amounts of amyloid in the spleen. The mink AEF model is a suitable tool to study the development of AA amyloidosis in a species with a spleen containing both well-developed ellipsoids and marginal zone.     

High molecular weight glycosaminoglycans in AA type amyloid fibril extracts from human liver.

Glycosaminoglycans have previously been identified in extracts of AA type hepatic amyloid fibril from a patient with amyloidosis associated with juvenile rheumatoid arthritis. The macromolecular properties of these polysaccharides are described here in more detail. By gel filtration and ion exchange chromatography glycosaminoglycans in the form of high molecular weight free chains were shown to coisolate with water extracted amyloid fibrils. About 60% of these were characterised as galactosamines (chondroitin sulphate/dermatan sulphate), whereas the remaining 40% consisted of N-sulphated glucosamines (heparin/heparan sulphate). The amyloid associated glycosaminoglycans were not part of intact proteoglycans in the fibril extracts.     

Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.     

Acceleration of murine amyloidosis by implantation of amyloid-containing grafts

We examined the transmissibility of amyloidosis by the implantation of amyloid-containing tissue. If the transmissibility similar to prion diseases is applicable, using amyloid-containing tissue for transplantation in humans might be a risk factor. In this study, AA amyloidosis occurred in mice that underwent implantation of AA amyloid-containing grafts to the liver and subsequent inflammatory stimulation. AApoAII amyloidosis occurred after implantation of AApoAII amyloid-containing grafts to the liver or to the subcutaneous space without inflammatory stimulation. Both types of amyloidoses occurred in the recipient mice sooner than expected. Moreover, AA and AApoAII amyloid deposits were found at 12 weeks after implantation in mice given AApoAII amyloid-containing grafts and inflammatory stimulation. These results suggest that implanted amyloid deposits have an AEF effect and that implanted amyloid-containing tissue can promote and accelerate a different type of amyloidosis. In another experiment, mice received amyloid-containing or normal tissue grafts. The degree of amyloid deposition was compared after 6 days and 5 weeks of inflammatory stimulation and when the mice were killed. There was no obvious difference in the degree of amyloid deposition between each group, indicating that the lag-time is shortened by implantation of amyloid-containing tissue, resulting in severe amyloidosis in the short term.     

Increased propensity for amyloidogenesis in male mice

BACKGROUND: The male sex is a risk factor for reactive amyloidogenesis in several disease entities. Environmental, socioeconomic or genetic factors may underlie this male preponderance. This study was aimed at discovering whether male sex predisposes to reactive amyloidosis also in mice and to elucidate some of the hormonal associations of this risk. METHODS: Male and female Swiss mice were subjected to an established amyloid induction protocol and the amount of their splenic amyloid was determined and compared. The effect of estrogen, progesterone, testosterone and adrenalin on amyloidogenesis was studied in both sexes by administering these hormnones during amyloid induction and comparing the amount of splenic amyloid of the study mice with the control mice which received the amyloid induction protocol alone. RESULTS: Amyloid deposition appeared to be more abundant in male mice. This gender difference was not associated with any of the 3 sex hormones tested. Despite an expected increment, adrenalin caused an attenuation of amyloid deposition. CONCLUSIONS: The preferential expression of reactive amyloidosis in male mice seems to be unrelated to the common sex hormones. Increased production of other hormones such as adrenalin, or perhaps an augmented susceptibility to their effect, may cause gender differences by suppressing female amyloidogenesis. Our study favors the hypothesis of genetic predisposition as the mechanism leading to sex differences in amyloidogenesis. Further validation of our findings in gonadal ablated models and other amyloid induction protocols is warranted.     

Urinary protein excretion patterns in reactive (secondary) systemic amyloidosis

Summary The urinary excretion of immunoglobulin light chains kappa and lambda, immunoglobulin G, transferrin, and beta-2-microglobulin was studied in 21 patients with nonimmunoglobulin-related amyloid nephropathy (secondary, type AA) associated with rheumatic disease and in 39 patients with glomerulopathy of nonamyloid origin, as well as in 22 patients with rheumatic disease without signs of nephropathy and in 15 healthy subjects. Patients with amyloidosis were found to have a higher ratio of excreted lambda/kappa light chains than patients with diabetic nephropathy or chronic glomerulonephritis. The increased lambda/kappa ratio was not dependent on the grade of proteinuria and was evident in patients with mild as well as heavy proteinuria. The ratio of lambda/kappa light chains in serum of patients with amyloidosis did not differ from that in healthy controls. The results suggest that amyloid deposition in the kidneys is associated with a selective alternation of the immunoglobulin light chain excretion in the urine.