Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.     

Colocalization of apolipoprotein AI in various kinds of systemic amyloidosis.

Apolipoprotein AI (apoAI), a major component of high-density lipoproteins, is one of the major amyloid fibril proteins and a minor constituent of the senile plaques observed in Alzheimer's disease. We examined colocalization of apoAI in various kinds of systemic amyloidosis in this study. Forty-three of 48 formalin-fixed paraffin-embedded heart specimens with various forms of systemic amyloidosis reacted immunohistochemically with anti-human apoAI antibody. ApoAI was also detected in water-extracted amyloid material by immunoblotting. In addition, we observed colocalization of apoAI and murine amyloid A (AA) amyloidosis in human apoAI transgenic mice. This is the first report of colocalization of apoAI with amyloid deposits in various forms of human systemic amyloidosis and murine AA amyloidosis in human apoAI transgenic mice. ApoAI may not always be a major component of amyloid fibrils, even when it is present in systemic amyloid deposits.     

Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation

Inflammation-related (AA) amyloidosis is a severe clinical disorder characterized by the systemic deposition of the acute-phase reactant serum amyloid A (SAA). SAA is normally associated with the high-density lipoprotein (HDL) fraction in plasma, but under yet unclear circumstances, the apolipoprotein is converted into amyloid fibrils. AA amyloid and heparan sulfate (HS) display an intimate relationship in situ, suggesting a role for HS in the pathogenic process. This study reports that HS dissociates SAA from HDLs isolated from inflamed mouse plasma. Application of surface plasmon resonance spectroscopy and molecular modeling suggests that HS simultaneously binds to two apolipoproteins of HDL, SAA and ApoA-I, and thereby induce SAA dissociation. The activity requires a minimum chain length of 12-14 sugar units, proposing an explanation to previous findings that short HS fragments preclude AA amyloidosis. The results address the initial events in the pathogenesis of AA amyloidosis.     

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.     

Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.     

Systemic AA amyloidosis in the red fox (Vulpes vulpes)

Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full‐length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter‐species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model.     

Immunocytochemical identification of amyloid in formalin-fixed paraffin sections

Summary Formalin-fixed paraffin sections of livers, spleens and kidneys from patients with primary, secondary and familial amyloidosis as well as from a casein-induced murine amyloid model were analysed by an immunocy-tochemical (unlabeled antibody enzyme) method utilizing antisera to amyloid-related proteins. All amyloid deposits of all amyloid types showed positive reactions with anti-AP of the respective species. Positive reaction of anti-human AA to human secondary amyloid deposits and of anti-mouse AA to the deposits of casein-induced murine amyloid was also observed, but there was no species cross reactivity. No significant deposition of the reaction products was produced by anti-immunoglobulin light chains on deposits of any amyloid type, or by anti-AA in the tissues from primary or familial amyloidosis. The results indicate that amyloid proteins AA and AP can survive as antigens through routine histologic preparation, that anti-AP can be a universal marker for deposits of any amyloid type within the same species, and that AA-type amyloid can be identified by this method while there may as yet be no feasible universal marker for the AL-type at present.Presented in part at the 64th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April, 1980     

Amino acid sequence variations in protein AA of cats with high and low incidences of AA amyloidosis

1. Amyloid isolated from the liver of a domestic short-haired (DSH) cat was dissolved and purified by gel filtration for amino acid sequence analysis. 2. Sequences of two major peptides corresponding to positions 18-23 and 25-75 of human amyloid protein AA were obtained when cyanogen bromide-cleaved protein was applied to an amino acid sequenator. 3. Comparison of these regions of amyloid protein from the Abyssinian cat (high incidence of AA amyloidosis) and DSH cat (low incidence of AA amyloidosis) revealed three amino acid differences, two of which occurred within regions that are completely conserved in the Abyssinian cat and all other species. 4. Secondary prediction plots showed less potential for amyloidogenicity (i.e., less beta-sheet conformation) in protein AA of the DSH cat as compared to the Abyssinian cat and other animal species. 5. These differences in protein AA of the DSH cat may, therefore, be linked to the comparatively uncommon occurrence of AA amyloidosis in the DSH cat as compared to the Abyssinian cat and other animals species.     

Senile cardiac amyloid: demonstration of a unique fibril protein in tissue sections.

Antisera were raised against degrading amyloid fibrils isolated from the heart of a patient with senile cardiac amyloidosis (SCA), and from a medullary carcinoma of the thyroid (MCT). The antisera were absorbed and used in indirect immunofluorescence to identify an amyloid fibril protein (ASCA) in heart tissue from patients with senile cardiac amyloidosis and to identify the amyloid fibril protein (AMCT) found in association with medullary carcinomas of the thyroid. Absorbed anti-ASCA antiserum did not react with normal tissue such as heart, liver, spleen, and striated muscle, or with amyloid tissue known to contain amyloid fibril proteins AA, AlambdaI, AlambdaIV, AlambdaV, AMCT or with pancreatic tissue containing islet amyloid deposits. The reactions with senile amyloid he,rt tissue could be blocked completely by degraded amyloid fibrils extracted from senile amyloid heart tissue or by amyloid fibril protein ASCA isolated from such fibrils. The anti-AMCT antiserum showed a similar specific reaction restricted to amyloid associated with MCT. In addition, antisera specific for amyloid fibril proteins AA, AlambdaI, AlambdaIV, and AlambdaV failed to react with senile cardiac amyloid, pancreatic islet amyloid, or medullary thyroid amyloid.     

Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

Interleukin 6-producing malignant mesothelioma

Systemic amyloidosis of the amyloid A (AA) type, is occasionally associated with various neoplasms, but the cause is still unclear. We obtained interleukin 6 (IL-6)-producing cells designated YO from a primary culture of a malignant peritoneal mesothelioma of epithelial type obtained from a 62-year-old woman. Post mortem examination revealed that the patient had systemic amyloidosis of the AA type. The supernatant media of YO cells, as well as recombinant human IL-6, successfully induced nonneoplastic liver cells to produce serum AA (SAA). Our data suggest that IL-6 produced by the tumor cells may have played an important role in the paraneoplastic syndrome of AA amyloidosis in this patient.     

Immunoelectron microscopic classification of amyloid in renal biopsies

Recent classification of amyloidosis is based on the chemical type of amyloid protein involved. In this study, routinely embedded kidney biopsies from nine patients with generalized amyloidosis and renal involvement were tested by immunoelectron microscopy, using the protein A-gold technique, with a panel of antibodies against the following amyloid proteins: AA, A lambda, A kappa and AF. Among the antibodies, the anti-AA was monoclonal (mc1) and the others polyclonal. In all nine cases, only one type of antibody reacted with each amyloid type. Six cases were classified as AA and three cases as A lambda type. These classifications were in agreement with the clinical data and the results of serum and urine immunoelectrophoresis. The gold particles were always associated with amyloid fibrils. No reaction was evident when an amyloid type was stained by a non-corresponding antibody, or in the four control cases without amyloid. The results show that antigenic classification of amyloid is feasible on routinely processed ultra-thin epoxy sections by immunoelectron microscopy, and thus affords the possibility of retrospective studies.     

The induction of accelerated murine amyloid with human splenic extract. Probable role of amyloid enhancing factor

Amyloid enhancing factor (AEF) is derived from the tissues of pre-amyloidotic and amyloidotic animals and, when transferred, greatly accelerates amyloid induction in the recipient murine models. It has also been reported that similarly accelerated amyloid induction can be achieved in mice by injection of human splenic homogenates from patients with amyloidosis. The present study has attempted to characterize further the mechanism of this "heterologous transfer of amyloid". Treatment of mice with the "tissue homogenate" or the "AEF extract" of AA-, AL- and A prealbumin-laden human spleens followed by daily subcutaneous casein injections induced amyloidosis in an accelerated fashion. The resultant amyloid deposits in mice had strongly positive immunohistochemical reactions with anti-mouse AA, and negative reaction with anti-human AA or anti-human prealbumin. The results lend support to the idea that accelerated amyloid induction in the recipient mice is unlikely to be due to transfer of human amyloid substance, but rather to formation of "native" murine amyloid under the influence of a human AEF factor similar to or identical with AEF described in mouse-to mouse transfer models.     

Accelerated resolution of AA amyloid in heparanase knockout mice is associated with matrix metalloproteases

AA-amyloidosis is a disease characterized by abnormal deposition of serum A amyloid (SAA) peptide along with other components in various organs. The disease is a complication of inflammatory conditions that cause persistent high levels of the acute phase reactant SAA in plasma. In experimental animal models, the deposited amyloid is resolved when the inflammation is stopped, suggesting that there is an efficient clearance mechanism for the amyloid. As heparan sulfate (HS) is one of the major components in the amyloid, its metabolism is expected to affect the pathology of AA amyloidosis. In this study, we investigated the effect of heparanase, a HS degradation enzyme, in resolution of the AA amyloid. The transgenic mice deficient in heparanase (Hpa-KO) produced a similar level of SAA in plasma as the wildtype control (Ctr) mice upon induction by injection of AEF (amyloid enhancing factor) and inflammatory stimuli. The induction resulted in formation of SAA amyloid 7-days post treatment in the spleen that displayed a comparable degree of amyloid load in both groups. The amyloid became significantly less in the Hpa-KO spleen than in the Ctr spleen 10-days post treatment, and was completely resolved in the Hpa-KO spleen on day 21 post induction, while a substantial amount was still detected in the Ctr spleen. The rapid clearance of the amyloid in the Hpa-KO mice can be ascribed to upregulated matrix metalloproteases (MMPs) that are believed to contribute to degradation of the protein components in the AA amyloid. The results indicate that both heparanase and MMPs play important parts in the pathological process of AA amyloidosis.     

Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein.

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.     

Widespread occurrence of AP in amyloidotic tissues. An immunohistochemical observation

Plasma (P)-component of amyloid (AP or SAP), while not an integral part of the amyloid fibril, has been considered to be intimately associated with virtually every different type of amyloid. In the present study, we evaluated the distribution of AP in the organs frequently involved in two forms of human systemic amyloidosis (AA and AF) and in mouse AA amyloidosis, by use of immunohistochemistry with anti-AP. Although the amyloid deposits generally showed moderate reactions with anti-AP, they were not always clearly distinguished from the surrounding non-amyloid tissue elements which often stained as well. The basement membrane often showed even stronger reaction to anti-AP than the adjacent amyloid deposits, and liver sections demonstrated such a high overall reaction to anti-AP that the anti-AP reaction on the amyloid deposits was often obscurred. The present results suggest that the binding between AP and the amyloid fibril may not be monospecific, that AP by this technique occurs rather widely throughout the body, and therefore that anti-AP may not be considered as specific a marker for amyloid deposits in immunohistochemical and perhaps other studies as well.     

The heterogeneity of protein AA in secondary (reactive)systemic amyloidosis

In secondary systemtic amyloidosis, amyloid fibrils have protein AA as a main subunit protein. As judged from gel chromatography and electrophoresis, this protein is rather homogeneous. In the present paper it is shown, however, that protein AA is very heterogeneous and composed of many peptides with different isoelectric points. However, their antigenic properties and amino acid compositions vary only little. It is concluded that protein AA is as heterogeneous as its postulated precursor, the acute phase reactant serum AA and that a theory that only one or a few serum protein AA's can give rise to amyloid fibrils, might be wrong.     

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4 °C, but were unstructured at 37 °C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37 °C, as evidenced by the increased thioflavin T (ThT) fluorescence and β-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4 °C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.     

AA-amyloidosis in dogs: partial amino acid sequence of protein AA and immunohistochemical cross-reactivity with human and cow AA-amyloid

Protein AA was purified from the kidneys of dogs with spontaneous reactive amyloidosis. The protein had a blocked N-terminal. Sequence analysis of a peptide obtained after cyanogen bromide cleavage revealed an amino acid sequence corresponding to positions 24-42 of human AA. This region showed a strong homology to protein AA of other species. Antiserum to both human and dog protein AA reacted immunohistochemically with AA amyloid of human, dog and cow origin.