Historical and Current Concepts of Fibrillogenesis and In vivo Amyloidogenesis: Implications of Amyloid Tissue Targeting.

Historical and current concepts of in vitro fibrillogenesis are considered in the light of disorders in which amyloid is deposited at anatomic sites remote from the site of synthesis of the corresponding precursor protein. These clinical conditions set constraints on the interpretation of information derived from in vitro fibrillogenesis studies. They suggest that in addition to kinetic and thermodynamic factors identified in vitro, fibrillogenesis in vivo is determined by site specific factors most of which have yet to be identified.

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.

Acute-phase serum amyloid A: perspectives on its physiological and pathological roles.

Serum amyloid A (SAA), a protein originally of interest primarily to investigators focusing on AA amyloidogenesis, has become a subject of interest to a very broad research community. SAA is still a major amyloid research topic because AA amyloid, for which SAA is the precursor, is the prototypic model of in vivo amyloidogenesis and much that has been learned with this model has been applicable to much more common clinical types of amyloid. However, SAA has also become a subject of considerable interest to those studying (i) the synthesis and regulation of acute phase proteins, of which SAA is a prime example, (ii) the role that SAA plays in tissue injury and inflammation, a situation in which the plasma concentration of SAA may increase a 1000-fold, (iii) the influence that SAA has on HDL structure and function, because during inflammation the majority of SAA is an apolipoprotein of HDL, (iv) the influence that SAA may have on HDL's role in reverse cholesterol transport, and therefore, (v) SAA's potential role in atherogenesis.

Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis.

Heparan sulfate promotes the aggregation of HDL-associated serum amyloid A: evidence for a proamyloidogenic histidine molecular switch.

During inflammatory diseases, serum amyloid A (SAA), an acute-phase apolipoprotein of HDL, can assemble into tissue deposits called AA amyloids. The mechanism and physiological factors promoting amyloidosis are largely unknown but likely involve heparan sulfate (HS), a glycosaminoglycan colocalized with all types of amyloids. In this study, we explored HDL-SAA:HS interactions using in vitro and cell culture assays to identify HS-binding domains that promote the conversion of native SAA into AA amyloid.

Secretion of amyloidogenic gelsolin progressively compromises protein homeostasis leading to the intracellular aggregation of proteins.

Familial amyloidosis of Finnish type (FAF) is a systemic amyloid disease associated with the deposition of proteolytic fragments of mutant (D187N/Y) plasma gelsolin. We report a mouse model of FAF featuring a muscle-specific promoter to drive D187N gelsolin synthesis. This model recapitulates the aberrant endoproteolytic cascade and the aging-associated extracellular amyloid deposition of FAF.

Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis.

Aims/hypothesis: Islet amyloid in type 2 diabetes contributes to loss of beta cell mass and function. Since islets are susceptible to oxidative stress-induced toxicity, we sought to determine whether islet amyloid formation is associated with induction of oxidative stress.

Methods: Human islet amyloid polypeptide transgenic and non-transgenic mouse islets were cultured for 48 or 144 h with or without the antioxidant N-acetyl-L: -cysteine (NAC) or the amyloid inhibitor Congo Red.

Serum amyloid A, in vivo splenic cholesterol export and its potential implications in hemolytic disorders.

A model to examine the in vivo relationship of acute phase serum amyloid A (SAA) to spleen cholesterol mobilisation was devised. Reticuloendothelial cells in vivo were loaded with a known quantity of cholesterol (1.5 mg) by infusing fragmented red blood cell membranes, which consist of approximately 50% cholesterol by dry weight.

Acute-phase-HDL remodeling by heparan sulfate generates a novel lipoprotein with exceptional cholesterol efflux activity from macrophages.

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDLratioHS interactions we discovered that HS caused significant remodeling of AP-HDL.

The anti-atherogenic potential of serum amyloid A peptides.

Serum amyloid A (SAA) inhibits acyl coenzyme A cholesterol acyltransferase and enhances cholesterol esterase activities, shifting stored esterified cholesterol to free cholesterol (the exportable form). The SAA domains responsible for these enzyme-modifying properties have been identified. These peptides are sufficiently small to be synthetically prepared to GMP quality and used in vivo to alter the progression of aortic lipid lesions in models of atherogenesis, suggesting that they may be clinically useful.

Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS-406 and azaserine result in reduced islet amyloid formation in vitro.

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in approximately 90% of individuals with Type 2 diabetes and is associated with decreased islet beta-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet beta-cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs).

Heparan sulfate as a therapeutic target in amyloidogenesis: prospects and possible complications.

Amyloid formation in vivo is a much more complicated process than studies of in vitro protein/peptide fibrillogenesis would lead one to believe. Amyloidogenesis in vivo involves multiple components, some no less important than the amyloidogenic protein/peptides themselves, and each of these components, and its role in the pathogenetic steps toward amyloid deposition could, theoretically, be a therapeutic target. Herein we use the definition of amyloid as it was originally described, discuss the similarities and differences between amyloid in vivo and in vitro, address the potential role of the extracellular matrix in in vivo amyloidogenesis by focusing on a specific component, namely heparan sulfate proteoglycan, and describe studies illustrating that heparan sulfate is a valid target for anti-amyloid therapy.

Sulfated cyclodextrins inhibit the entry of Plasmodium into red blood cells. Implications for malarial therapy.

The effect of sulfated cyclodextrins on Plasmodium falciparum cultures was determined. alpha-, beta-, and gamma-Cyclodextrins having equal degrees of sulfation inhibited parasite viability to a similar degree, a result suggesting that the ring size of the cyclodextrin is not a critical factor for inhibitory activity. beta-Cyclodextrins containing fewer than two sulfate groups had no inhibitory activity, however, compounds containing 7-17 sulfates were found to be active in the microM range.

The path of murine serum amyloid A through peritoneal macrophages.

Serum amyloid A (SAA) is a family of proteins encoded by four related genes. Of the four, isoforms 1.1 and 2.

Rapid recycling of cholesterol: the joint biologic role of C-reactive protein and serum amyloid A.

Proteins that are highly conserved throughout evolution are presumed to have critical roles in the survival of the species. The two major acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) increase up to 1000-fold during inflammation. Both proteins have been highly conserved phylogenetically for at least the last 500 million years.

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro.

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[3H]glucosamine, [35S]sulfate, and L-[14C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-(3) and beta-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[3H]glucosamine, but not of [35S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[14C]leucine incorporation into total protein synthesis. These results suggest that analogs 3-5 exhibit an inhibitory effect on D-[3H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[3H]glucosamine and by competing for the same metabolic pathways.

Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesions in apoE-/- mice.

Macrophages (Mphi) at sites of acute tissue injury accumulate and export cholesterol quickly. This metabolic activity is likely dependent on the physiological function of a major acute-phase protein, serum amyloid A 2.1 (SAA2.

Preparation and propagation of amyloid-enhancing factor.

Amyloid-enhancing factor (AEF) is a biological "activity" that is defined in the context of inflammation-associated amyloidogenesis (AA). When administered intravenously to mice followed by an inflammatory stimulus, such primed mice deposit substantial AA amyloid in spleen within 36-48 h. Since experimental induction of AEF is dependent on amyloidogenic protocols, and rapid AA amyloid induction is dependent on AEF, a strategy for AEF isolation is required to break into this circular process.

In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis.

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction.

Amyloidogenesis recapitulated in cell culture: a peptide inhibitor provides direct evidence for the role of heparan sulfate and suggests a new treatment strategy.

To date 22 different polypeptides, including Abeta in Alzheimer's disease and PrP(Sc) in prion disorders, are known to re-fold and assemble into highly organized fibrils, which associate with heparan sulfate (HS) proteoglycans to form tissue deposits called amyloid. Mononuclear phagocytes have long been thought to be involved in this process, and we describe a monocytic cell culture system that can transform the acute-phase protein serum amyloid A (SAA1.1) into AA-amyloid and appears to recapitulate all the main features of amyloidogenesis observed in vivo.

Novel glycosaminoglycan precursors as antiamyloid agents: Part IV.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, beta-amyloid (Abeta), in Alzheimer's disease. The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere.

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.

A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein: significance for malarial sporozoite attachment to hepatocytes.

Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP.

Novel glycosaminoglycan precursors as anti-amyloid agents, part III.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, e.g.

Macrophage cholesterol efflux and the active domains of serum amyloid A 2.1.

Serum amyloid A 2.1 (SAA2.1) suppresses ACAT and stimulates cholesteryl ester hydrolase (CEH) activities in cholesterol-laden macrophages, and in the presence of a cholesterol transporter and an extracellular acceptor, there is a marked increase in the rate of cholesterol export in culture and in vivo.