Medin amyloid - a matter close to the heart172508/FULLTEXT01.pdfList of papers This thesis is based...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 374

Medin amyloid - a matter close tothe heart

Studies on medin amyloid formation andinvolvement in aortic pathology

ANNIKA LARSSON

ISSN 1651-6206ISBN 978-91-554-7277-1urn:nbn:se:uu:diva-9275

�� !�� "�� # ��$% &� �'� �� & �� & ('��'� )!�� & *��+, -'� �� .�� /��',

��

0�� 1, � #, *�� 2 � �� '� '��, 3�� &�� '��, 1�� , �� $45, 64��, ��, 7389 :4#2:�266524�442�,

1�� & �� &�� '��;�� & ��&�� , *�� .'��' �� '� &�� & �'�� '�� '�� & �'� �'�� 6 ��, -'� &�� &�� 6 �� &�� & �'� �� '��, �. �� .� �� 7 .� �� .��' ��'��'�� '�� ' ��'�� .��' �� '�� '�� &�� &��, 7 �� 77� .� &��'�� '.�� '�� '� �� #2�:�� '� ��2�� ,7 �� 777� �'� ��<�� & �� .�� , 1�� &��

�� .��' �'�� .�� &� �� , -'�� &�� '�� '� �. �� '�� , 7�� '�� '� �� & �� &�� '�� '� � �� &��, 7 �� & �'��&�� .� �� '�� &�� &�� .�� ,1�� &�� 2�� , 1�� & ��&�� &��

�� &�� '�� ,7 �� 7=� �� 12�� )11+ �� .�� &�� 2��;�� .��' �� '� ��, 7 �� &�� '�� '� &�� & 11 &�� &� �� '��, -'� �� & �� '�� '�� &�� & �� &�� & ��'�� ,7 �� '� �� & �'�� '�� '�� '� '. �� &�� '� &�� &

��'�� '� ��<�� & �� &� �� '��,

� �� 11 �� '�� '��

�� ! � �� " � �� #��! $��% ��%�� ! �� ! �&'()*+) ��! ��

> 1�� 0�� #

7339 �?6�2?� ?7389 :4#2:�266524�442��%�%��%��%��2:�46 )'��%@@��,��,��@��A��B��%�%��%��%��2:�46+

Till Johan & Hanna

List of papers

This thesis is based on the following papers, which are referred to in the text by their roman numerals (I-IV). The papers are appended at the end of the thesis. I Larsson A, Peng S, Persson H, Rosenbloom J, Abrams WR,

Wassberg E, Thelin S, Sletten K, Gerwins P and Westermark P. Lactadherin binds to elastin – a starting point for medin amyloid formation? Amyloid (2006) 13(2), 78-85

II Larsson A, Söderberg L, Westermark GT, Sletten K, Engström

U, Tjernberg LO, Näslund J and Westermark P. Unwinding fibril formation of medin, the peptide of the most common form of human amyloid. Biochemical and Biophysical Research Com-

munications (2007) 361, 822-828 III Larsson A*, Peng S*, Wassberg E, Gerwins P, Thelin S, Fu X

and Westermark P. Role of aggregated medin in the pathogenesis of thoracic aortic aneurysm and dissection. Laboratory Investiga-

tion (2007) 87, 1195-1205 IV Larsson A, Malmström S and Westermark P. Signs of cross-

seeding: role of aortic medin amyloid as a trigger for protein AA deposition. Manuscript

* These authors should be considered as joint first authors.

All published material is reproduced with permission from the publishers.

Contents

Introduction....................................................................................................... 11

Amyloid ............................................................................................................ 12 Protein folding and misfolding ................................................................... 12 The fibrillar component............................................................................... 13 Additional components ............................................................................... 14 Amyloid formation ...................................................................................... 15

Protein destabilization ............................................................................ 15 Fibril growth ........................................................................................... 17

Amyloid diseases ......................................................................................... 18 Definitions............................................................................................... 18 Consequences of amyloid deposition .................................................... 20

Therapies ...................................................................................................... 21 Reducing the precursor protein .............................................................. 21 Inhibition of fibril formation.................................................................. 21 Amyloid clearance .................................................................................. 22

Functional amyloid...................................................................................... 23 Amyloid methodology................................................................................. 23

Detection ................................................................................................. 23 Isolation and purification ....................................................................... 25 Characterization ...................................................................................... 25

Medin amyloid.................................................................................................. 27 Lactadherin - the precursor ......................................................................... 27

Structure .................................................................................................. 28 Function................................................................................................... 29

Medin prevalence and distribution ............................................................. 32 Medin and other vascular amyloid ............................................................. 34 Medin fibrillation......................................................................................... 35 Consequences of medin deposition ............................................................ 36

Thoracic aortic aneurysm and dissection .............................................. 36 Temporal arteritis.................................................................................... 37 Artery stiffening...................................................................................... 38

Concluding remarks and future perspectives.................................................. 39

Populärvetenskaplig sammanfattning ............................................................. 41

Acknowledgements .......................................................................................... 45

References......................................................................................................... 47

Abbreviations

AA amyloid protein A Aß amyloid ß-peptide AßPP amyloid ß precursor protein AL amyloid protein of immunoglobulin light chain origin ApoE apolipoprotein E APrP amyloid prion protein CAA cerebral amyloid angiopathy DAB 3,3’-diaminobenzidine tetrahydrochloride EGF epidermal growth factor ECL enhanced chemiluminiscence ELISA enzyme-linked immunosorbent assay FAP familial amyloidotic polyneuropathy GCA giant cell arteritis HRP horseradish peroxidase HS heparan sulfate IAPP islet amyloid polypeptide MFGM milk fat globule membrane MFG-E8 milk fat globule EGF 8 MMP matrix metalloproteinase RP-HPLC reversed phase-high performance liquid chromatography SAA serum amyloid A SAP serum amyloid P component SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis TAA thoracic aortic aneurysm TAD thoracic aortic dissection TTR transthyretin VEGF vascular endothelial growth factor

11

Introduction

The term amyloid is derived from the Greek word for starch, amylon, and was introduced in 1854 by Rudolph Virchow, as he believed the waxy ab-normalities observed at autopsy consisted of carbohydrates.1 However, only a few years later, in 1859, Friedreich and Kekulé demonstrated that amyloid aggregates are of protein origin.2 Amyloid proteins undergo conformational changes and self-assemble into stable insoluble amyloid fibrils, with a high degree of ß-sheet structure. To date, 28 proteins have been identified as amy-loidogenic in man,3,4 the best known ones being the prion protein (APrP) in Creutzfeldt-Jakob disease and the Aß protein precursor (AßPP) in Alz-heimer’s disease. The most prevalent type is medin amyloid, which arises from an internal fragment of the protein lactadherin.5 Medin amyloid is found in the thoracic aorta in almost all individuals above 50 years of age.6 Despite its high prevalence, the knowledge of medin amyloid has been lim-ited. In this thesis, different aspects of medin amyloid have been investi-gated, with the overall aim to elucidate its formation, involvement in aortic pathology and function of the precursor protein lactadherin.

12

Amyloid

Protein folding and misfolding Essentially all processes that constantly occur throughout our bodies rely on the interplay between proteins. In order to function properly a protein re-quires its correct conformation. The amino acid sequence determines the basic architecture of a protein. The unfolded peptide chain has the highest amount of free energy and many different possibilities to fold. As the folding process starts, intramolecular interactions form, the proteins become more organized and the amount of free energy decreases. The native protein is usually the conformation with the lowest amount of free energy and thus the most thermodynamically stable.7

In the crowded and rather rough environment of the cell there are many pitfalls on the way to a proper three-dimensional structure.8 To avoid mis-folding and aggregation of proteins, an elaborate folding machinery includ-ing chaperone and protease systems has evolved. Chaperones are a diverse group of proteins that facilitate correct protein folding and prevent non-native proteins from aggregation into non-functional structures. If a mis-folded protein persists it can be recognized and proteolytically degraded by various proteases. The proteasome, which is the major site for proteolysis in the cell, is a cytosolic protein complex that degrades misfolded proteins that have been tagged with a polyubiquitin chain.9,10 Most of the knowledge of the protein misfolding control system is from inside the cell. Very little is known about what happens to a non-native protein outside the cell. How-ever, intuitively such a system should exist extracellularly as well. Only recently two proteins, clusterin and haptoglobin, have been identified as chaperones that assist in the folding of misfolded proteins in the extracellular space.11-13

There are gaps in the defense against non-native proteins as these may es-cape the quality-control system. Misfolded proteins are prone to self-associate and may form pathological aggregates. The amyloidoses are exam-ples of diseases with misfolded protein infiltrates as histopathological hall-marks.

13

The fibrillar component The amyloid aggregate mainly consists of a fibrillar protein component, which is a highly ordered assembly, formed by the self-association of thou-sands of misfolded protein molecules that are hydrogen bonded into a stable cross-ß structure. For a long time knowledge of the amyloid structure was restricted due to limitations of the existing imaging techniques, predomi-nantly transmission electron microscopy and x-ray fiber diffraction. Informa-tion from more recent techniques, such as atomic force microscopy,14 nu-clear magnetic resonance15,16 and crystal x-ray diffraction analysis,17,18 have provided more details of the amyloid fibril structure.

In the amyloid fibril, the protein monomer harbors one or more stretches with ß-strand conformation. Numerous monomers are stacked on top of each other through hydrogen bonds, 4.7 Å apart, in a parallel or anti-parallel ar-rangement to form a ß-sheet structure along the length of the fibril. In turn, different ß-sheets interact at a distance of 10 Å, perpendicular to the fibril axis, to make up the protofilament (Figure 1), the underlying subunit of the amyloid fibril. In the amyloid fibril, which is non-branching and has a di-ameter of 7-12 nm, two to six protofilaments are aligned in parallel and often slightly twisted around each other.19-21

Figure 1. Schematic figure of a protofilament with the characteristic cross-� struc-ture. The protofilament is composed of two or more sheets 10 Å apart. Two to six protofilaments make up the amyloid fibril (not shown).

Even though the fibrils share these common structures there are certain differences attributable to the peptide sequence and its side chains. These variations include: 1) lengths of the polypeptide chain involved in the core structure and if arranged in a parallel or anti-parallel fashion, 2) features of loops, turns and other regions outside the core region, 3) the number of beta-

Peptide monomers (ß-strands)

ß-sheet(monomers 4.7 Å apart)

Protofilament(sheets 10 Å apart)

4.7 Å

10 Å

Fibr

il ax

is

14

sheets that constitute the protofilament and 4) the number of protofilaments in the fibrils.20

Amyloid fibrillation is a complicated process of which many aspects are still obscure and unresolved. In recent years there has been a lot of focus on early non-fibrillar aggregates, as these are believed to induce toxicity rather than the mature fibrils (discussed below).22-27 There is confusion regarding the terminology of these species and what they are. Many laboratories use their own definitions. They have been called many names, such as micelles, oligomers, protofibrils (as opposed to protofilaments, which are the struc-tural elements of the amyloid fibril) and spheroids, and their size has been estimated from a few up to thousands of monomers.23,26-28 The smaller ag-gregates are spherical in shape, whereas flexible fibre-like structures have been proposed for the larger protofibrils.21 It is still not clear whether these species are obligate intermediates on the way to mature amyloid fibrils (on-pathway) or if they follow a different aggregation pathway (off-pathway) distinct from the classical nucleation-dependent process described below.14,29 In this thesis, these smaller prefibrillar aggregates will be referred to as oli-gomers.

Additional components Although the main component of amyloid is the fibrillar protein, additional components are involved in the deposits.

Virchow’s observation in 1854 that amyloid consisted of carbohydrates was not completely wrong,1 as the glycosaminoglycans, particularly heparan sulfate (HS), are common in amyloid aggregates.30 HS increases the ß-sheet structure of the serum amyloid A (SAA), the protein that gives rise to AA amyloidosis.31 Furthermore, in mice there is an up-regulation of HS mRNA prior to AA deposition, implicating the importance of HS in early fibrillo-genesis.32 A HS binding site has been identified on the SAA molecule and it has been proposed as a potential target in the development of anti-AA amy-loid drugs.33 Heparanase fragments HS, and, in an interesting study on mice with heparanase over-expressed in all tissues except for the spleen, AA amy-loid developed only in the spleen.34 Although most of the HS-amyloid find-ings are from the AA amyloidosis field, HS interactions appear to be of im-portance in other types of amyloidoses. In vitro, HS binds to Aß and IAPP and accelerates the fibril formation of these peptides.35,36 In a murine Alz-heimer model, treatment with low molecular weight heparin (closely related in structure to HS) reduced the Aß plaque load and attenuated Aß-mediated toxicity in cell culture. The mechanism of action is not clear, but the drug may work as a HS analogue that prevents the HS-Aß interaction.37

Other extracellular components, such as collagen IV, laminin and fi-bronectin, have been observed co-localized to various types of amyloid de-

15

posits.38,39 Like HS, these extracellular proteins are believed to facilitate fibrillation by functioning as a scaffold onto which numerous monomers can bind and they may thereby also determine the tissue localization of amyloid aggregates.40

In addition, all amyloid deposits investigated also contain serum amyloid P component (SAP).41,42 SAP, which is a pentameric protein where each monomer is 25 kDa, binds to specific conformations common to all amyloid fibrils and is thought to stabilize the amyloid fibrils and to protect against proteolysis.43 Mice with the SAP gene knocked out develop amyloidosis much later than mice expressing the protein, suggesting that SAP affects amyloidogenesis but is not a prerequisite for amyloid formation.44 The SAP-amyloid interaction is utilized in amyloid diagnostics. Radiolabeled SAP, which is injected intravenously into patients, binds to amyloid fibrils and enables imaging of aggregate size and localization.45

Apolipoprotein E (apoE) is another constituent of amyloid aggregates.46 However, its role there remains elusive. Interestingly, persons carrying one or two copies of the apoE-�4 allele have an increased risk of developing Alzheimer’s disease47,48 and ApoE-knockout mice have reduced Aß-amyloid load.49

Amyloid formation The formation of amyloid may be separated into two critical events, protein destabilization and nucleation.

Protein destabilization

To date 28 proteins have been identified to form amyloid in man and the identity of many more remains to be determined.3,4 The amyloid proteins are a diverse group of proteins with no general similarity, although the apolipo-proteins and polypeptide hormones are over-represented. Because of this diversity and the fact that many peptides, not associated with amyloid dis-eases, form amyloid-like fibrils in vitro, it has been proposed that all poly-peptide chains have a generic ability to convert into amyloid structures.50

Aggregation and fibril formation generally start as a result of destabilized structures (Figure 2), which can occur through several mechanisms depend-ing on protein. The Val30Met mutation in the transthyretin (TTR) gene de-stabilizes the native protein fold and makes it prone to aggregate, resulting in familial amyloidotic polyneuropathy (FAP).51 In some diseases, for example AA amyloidosis, high concentrations of the precursor protein are required for amyloid formation.52 Proteolytic cleavage of the precursor protein is a prerequisite for fibril formation during Alzheimer’s disease53 and gelsolin-derived amyloidosis.54 It is believed that interaction with certain tissue com-

16

ponents may be of importance for the fibrillation process. In dialysis-related amyloidosis, aggregates of ß2-microglobulin are found associated to collagen fibrils55 and deposits of medin amyloid are commonly observed adjacent to the elastic fibers of the aorta.6 Some amyloid proteins, for example the prion protein, contain stretches of amino acids with �-helical conformation that, according to secondary structure predictions, should form a ß-strand. Pro-teins harboring these so called discordant helices are more prone to unfold and aggregate.56 Many of the known amyloidoses are senile, i.e. they start to form during aging.3 Why this happens is not known, but may be a conse-quence of impaired chaperone-protease machinery. Inactivation of the tran-scription factor HSF-1 in Caenorhabditis elegans induces an aging pheno-type and reduces life span. HSF-1 promotes the transcription of heat-shock genes, including chaperones and proteases. Interestingly, hsf-1 RNAi-treated animals showed earlier aggregation of polyglutamine-repeat proteins (a characteristic of Huntington’s disease), thereby providing an explanation for the increase in protein aggregation during aging.57

Figure 2. A simplified view of amyloid formation.

Much of the information regarding amyloid fibrillation has been acquired from the test tube. Charge, ß-strand content and hydrophobic residues are peptide characteristics that influence fibril formation.58-60 Furthermore, the solvent used is important for fibrillation. Ionic strength, low pH, agitation and heat are parameters commonly used to destabilize proteins and speed up the formation of amyloid-like fibrils.20,61 Interestingly, it has been observed

Amyloid

Aggregation

Aggregation

Destabilization

Oligomer (toxic)

Cell death

Misfolded protein(non-functional)

Native protein (functional)

Apoptotic cell

17

that fibrils derived from the same peptide or protein may have different mor-phology as a result of the conditions under which fibrillization takes place.62

Fibril growth

Once the proteins are destabilized fibrillation can start. Amyloid fibril growth is a nucleation-dependent process and can be divided into two phases, the lag and the elongation phases. The lag phase involves the assem-bly of protein monomers into an oligomeric nucleus. This step is energeti-cally unfavorable and thus rate-limiting. Once the nucleus has formed the elongation phase, which is energetically favorable, starts and the fibrillation of the remaining protein is rapid. By adding preformed fibrils to an amyloi-dogenic protein solution it is possible to bypass the slow nucleation event and thereby accelerate the formation of soluble proteins into amyloid-like fibrils, a phenomenon termed seeding (Figure 3).63,64 Cross-seeding refers to seeding with heterologous fibrils, i.e. fibrils of a biochemical nature different from the protein to be seeded.65 The research on prion diseases, of which the mad-cow disease has attracted most attention in the last years, demonstrates that homologous seeding (from cow to cow) as well as heterologous seeding (from cow to human) also occur in vivo.66

Figure 3. Fibrillization of protein X with and without seed. In the presence of seed fibril formation is accelerated.

Experimental AA amyloidosis in mouse is a convenient model to study these phenomena in vivo and has provided many exciting details about the mechanisms of amyloid formation. AA amyloidosis can easily be induced in many mouse strains by inflammatory challenges, such as subcutaneous in-jections of silver nitrate. The time to develop amyloidosis is drastically shortened by co-injecting a small amount of AA amyloid fibrils.67 However, not only AA fibrils are potent in inducing amyloidogenesis. Fibrils of other

18

amyloid proteins68 as well as amyloid-like fibrils from proteins in nature, such as silk fibers,65 also have this capacity. Interestingly and somewhat frightening, this “transmissibility” of disease is also observed, although with reduced potency, when fibrils are administrated orally.67 Geese and ducks used for the production of foie gras are commonly forced fed in order to obtain large fatty livers. As a side effect these animals commonly develop AA amyloid in the liver. When the murine AA model was fed such liver paté amyloidosis developed at an increased rate.69

Amyloid diseases

Definitions

Amyloidoses are diseases where there is a component of amyloid histologi-cally. To be called amyloid certain criteria need to be fulfilled. The deposits have to occur in vivo, show typical x-ray diffraction pattern, induce green birefringence in polarized light when stained with Congo red and show straight and unbranched fibrils in the electron microscope. Earlier the defini-tion of amyloid required the deposits to be extracellularly. But given that amyloid formation, in some cases, is thought to be initiated inside cells and the existence of intracellular substance with characteristic amyloid structure, the definition was changed to include intracellular inclusions as well.3 This means that intracellular aggregates of the Tau-protein, which form during Alzheimer’s disease in parallel to extracellular Aß-amyloid, now are re-garded as amyloid. There are amyloid proteins that still have not been con-nected to any disease, such as prolactin-derived amyloid in aging pituitaries and semenogelin I-derived amyloid in vesicula seminalis.3 In some cases there is a disease connected with an amyloid component but the amyloid protein has not been identified. To this group belong the amyloid compo-nents of calcified cardiac valves70 and basal cell carcinoma.71

The amyloidoses can be divided into localized or systemic forms, depend-ing on deposition. Type II diabetes, Alzheimer’s disease and Creutzfeldt-Jakob disease are all localized amyloidoses, i.e. only one organ is affected by amyloid infiltrates. In diabetes, the islet amyloid polypeptide (IAPP) depos-its as amyloid in the pancreas, whereas in Alzheimer’s and in Creutzfeldt-Jakob diseases the Aß peptide and the prion protein, respectively, form amy-loid in the brain. More uncommon diseases like AA and light chain (AL) amyloidoses are classified as systemic forms since the involved proteins deposit in a wide range of tissues and organs. In general the systemic amy-loidoses develop from circulating plasma proteins, whereas the localized forms are derived from precursor proteins expressed at the site of deposition. See Table 1 for an overview of the different localized and systemic amyloi-doses and the corresponding amyloidogenic protein.3,4

19

Amyloid Protein

Precursor Protein Localized (L) or Systemic (S)

AA (Apo)serum AA S

AApoAI Apolipoprotein AI S, L

AApoAII Apolipoprotein AII S

AApoAIV Apolipoprotein AIV S

A�2M �2-microglobulin S

ABri ABriPP S

ACys Cystatin C S

AFib Fibrinogen �-chain S

AGel Gelsolin S

AH Immunoglobulin heavy chain S, L

AL Immunoglobulin light chain S, L

ALys Lysozyme S

ATTR Transthyretin S

AANF Atrial natriuretic factor L

A� A� protein precursor (A�PP) L

ACal Calcitonin L

ADan ADanPP L

AIAPP Islet amyloid polypeptide L

AIns Insulin L

AKer Kerato-epithelin L

ALac Lactoferrin L

ALECT2 Leucocyte chemotactic factor 2 L

AMed Lactadherin L

AOaap Odontogenic ameloblast-associated protein L

APrP Prion protein L

APro Prolactin L

ASemI Semenogelin I L

ATau Tau L

Table 1. The 28 known amyloidogenic proteins in man and the corresponding pre-cursor protein.3,4

20

Consequences of amyloid deposition

The symptoms of amyloidoses differ between the amyloid diseases and de-pend on the tissue distribution of amyloid and/or oligomeric aggregates. In Alzheimer’s disease there is cognitive decline because of aggregates in the brain. Within FAP patients, aggregates are initially found in the peripheral nerves, resulting in polyneuropathy. In AL amyloidosis, which is the most heterogenous disease, amyloid can be found in virtually any tissue and thus cause a wide range of symptoms, such as heart failure, atrophy of hand mus-cles, gastrointestinal bleedings, nephrotic syndrome, macroglossia and weight loss.40,72 This makes it difficult for the clinician to make a correct diagnosis in time for an efficient treatment.

The pathology of amyloidoses may be attributed to the large amounts of amyloid that may be deposited. Particularly in systemic amyloidoses, kilo-grams of amyloid may fill up an organ, thereby heavily affecting its func-tion. Many of the systemic amyloidoses are very serious and have a quick disease progression. Unfortunately, there is still no efficient treatment of these diseases, and many patients die within a couple of years after diagno-sis.40,72,73 However, an alternative and widely accepted theory suggests that the toxicity of the protein aggregates is the primary cause of pathogenesis, especially in the localized amyloidoses, which may contain only small amounts of amyloid. In clinical data from Alzheimer patients there is often poor correlation between amyloid load and disease progression.74,75 A patient could be severely demented but show very small amounts of Aß-amyloid plaques. In general, the cognitive decline is better correlated to smaller preaggregates or Aß oligomers.75 Similar observations have been made for patients with TTR amyloidosis. Symptoms of disease start to appear before amyloid has developed, when only small aggregates are present. The aggre-gates cannot be detected with the amyloid-specific stain Congo red, but can be seen with a TTR-specific antibody.24 Because of these and many more findings it has become evident that it is not the mature amyloid fibrils that are toxic but rather earlier oligomeric assemblies. Also, experimental data from animal models26,76,77 and cell culture studies22,23,25,78 identify the oli-gomers as the most pathogenic species.

A large portion of today’s amyloid research is focused on the toxic mechanisms of amyloid oligomers. In the oligomeric non-native state, resi-dues are exposed on the surface that in the native conformation is buried on the inside of the protein. Inside the crowded milieu of the living organism, interactions between oligomers and cellular as well as extracellular compo-nents will take place, which may result in events not normally occurring in healthy tissue. Numerous reports show how amyloid oligomers in many different ways affect the cellular machinery, ultimately leading to cell death. These include induction of oxidative stress,79-81 membrane disruption or for-mation of pore-like structures in the cell membrane,23,82,83 and mitochondrial

21

dysfunction.84,85 Interestingly, in vitro-formed oligomers of proteins, not normally associated with amyloidosis, also induce toxicity in the same fash-ion as true amyloid oligomers.86

Given the growing body of evidence that amyloid oligomers are the most deleterious species, it has been proposed that the amyloid fibril could be a protection state that is formed to shield the cells from oligomers.87 In support of this theory, it was recently demonstrated that Sis1, a Hsp40 chaperone, promoted amyloid fibrillization of the yeast prion and thereby protected the cells from toxic oligomeric species.88 Thus, attempts to develop therapies based on breaking up the amyloid fibril could have aggravating effects, as the levels of toxic species may be increased.

Therapies In the last decade the knowledge of amyloid formation and its components has lead to the development of many new alternative therapies. While exist-ing treatments try to reduce the amounts of the precursor protein, the more recent approaches aim at inhibiting fibril formation and clearing amyloid deposits.

Reducing the precursor protein

Current treatments of amyloid diseases are mainly based on reducing the supply of amyloid precursor protein as well as supporting the function of the affected organs, which may involve organ transplantation. In AL amyloido-sis, where there is an underlying B-cell dyscrasia, chemotherapy is used to arrest the production of amyloidogenic light chains.89 Persons suffering from familial amyloid polyneuropathy may have one of over 100 known muta-tions with a destabilizing effect on the TTR protein, rendering it amyloi-dogenic. TTR is mainly produced by the liver and liver transplantations are therefore performed in order to halt disease progression.90 AA amyloidosis is treated with anti-inflammatory drugs since these suppress the chronic in-flammation and thereby reduce the amount of the precursor SAA, which is an acute phase protein.91

Inhibition of fibril formation

There are several novel therapeutic approaches with the goal to prevent fibril formation. One such approach aims at stabilizing the precursor protein and has mainly been investigated in the TTR field. TTR fibrillization involves an initial dissociation of its tetramer structure into single monomers. With small molecules that interact with the TTR molecule it has been possible to stabi-lize the tetrameric form of the protein, thereby preventing amyloid forma-

22

tion. One such molecule is the anti-inflammatory agent diflunisal, which has shown promising results. The compound stabilizes the structure of mutated TTR in transgenic mice92 and non-mutated TTR in human controls.93 No studies are yet published on the effects of diflunisal on human patients. However, a large international study on FAP patients is currently ongoing.

Attempts to target heparan sulfate have been undertaken in order to in-hibit AA fibril formation. Eprodisate, which is a small molecular analogue of heparan sulfate, has gone through clinical trials of 183 patients with AA amyloidosis. Patients treated with the drug showed a reduced decline of re-nal function compared to placebo-treated patients.94

The Aß peptide is formed when the Aß precursor protein is cleaved by two enzyme complexes, the ß- and the �-secretases. When residing in the precursor protein the peptide is in its native conformation, but as it is cleaved it becomes destabilized and amyloidogenic. Ongoing research is trying to identify drugs that block the two secretases in order to reduce the formation of the peptide.95

Amyloid clearance

Immunotherapy is an appealing therapeutic approach to reduce amyloid load. During passive immunization synthesized components of the immune system are administrated to a person; hence there is no need for the body’s own immune system to produce these components. An antibody that recog-nizes a generic conformation found on different amyloid fibrils has been generated.96 When transferred to mice with AL amyloidosis as well as to mice with AA amyloidosis, amyloid burden was reduced through the release of proteolytic enzymes by activated neutrophils. The antibody has been chi-merized and prepared for eventual phase I and II clinical trials on patients with AL amyloidosis.97,98 Furthermore, an antibody that is claimed to interact only with oligomeric forms of amyloid proteins has been developed and may serve an important role in future amyloid therapeutics.99

During active immunizations, an antigen is injected into a person and the body itself generates the immunity towards the antigen, through activation of T and B cells. Aggregated Aß has been used for vaccination of Alzheimer’s disease patients, but the study was interrupted due to development of menin-goencephalitis in some patients. However, the study presented some promis-ing data as patients who generated Aß antibody titers showed a decreased cognitive decline compared to those without antibody response.100

SAP binds to all amyloid fibrils and is thought to protect the fibrils from degradation by proteolytic enzymes and phagocytic cells.43 A compound shortened CPHPC has been developed that crosslinks two SAP pentamers, whereby the binding face is blocked and the SAP-amyloid interaction is inhibited. By reducing SAP levels it is believed that the amyloid deposits will be destabilized and prone to regress. In addition, as the serum levels of

23

SAP decrease, new amyloid deposition is prevented.101 Clinical studies are now underway to investigate the efficacy of CPHPC in patients with sys-temic amyloidoses.

The antibiotic doxycycline has also been investigated as a potential drug against amyloidosis. In vitro as well as in a FAP transgenic mice model, doxycycline treatment resulted in complete disaggregation of fibrils, without increasing the levels of toxic oligomeric species.102,103 In amyloidosis the drug probably functions as a fibril disrupter. However, there are to date no reports of clinical trials with this compound.

Functional amyloid It has become evident that the amyloid structure is not merely involved in disease but has also been utilized by living systems for various purposes.

The interaction of Escherichia coli to inert surfaces and host proteins are mediated by curli, a class of surface proteins with a fibrillar cross-ß structure that bind the amyloid-specific dyes Congo red and thioflavin T.104 Another bacterium, Streptomyces coelicolor, uses the amyloid-like structure in aerial hyphae for efficient spread of spores.105

Functional amyloid has also recently been suggested in man. In the mela-nocytes, melanin is formed and assembled on a fibrous material with some characteristics of amyloid. The fibrillar protein has been identified as Pmel17. The instant fibrillation of Pmel17 and the localization to the mela-nosome compartment are suggested to mitigate the toxicity usually associ-ated with amyloid formation.106

These are a few examples of amyloid-like structures that have benevolent effects in organisms and show that nature has selected, or at least not select-ed against, some proteins with an amyloid structure. In the nanotechnology field the structure is even being mimicked in an attempt to create robust and biocompatible materials with diverse applications, from tissue engineering to nanoelectronics.107-109

Amyloid methodology

Detection

Various methods are available for amyloid detection. Congo red has been used to detect amyloid since 1922.110 Initially Congo red was intravenously injected and the plasma clearance of the dye was correlated to amyloid load. Persons with amyloid have a more rapid clearance since Congo red is drained from the plasma by the amyloid fibrils, with which it interacts. However, the method had some drawbacks and caused allergic reactions and

24

even death in some patients. Later the dye was used for staining of tissue sections and has since then been the most commonly used method for detec-tion of amyloid in tissue with light microscopy. In normal light, Congo red-stained amyloid is red (Figure 4a) but through crossed polars it becomes green or yellow (Figure 4b), normally described as apple-green birefrin-gence. It is not completely known how Congo red visualizes amyloid but it is believed to bind the characteristic cross-ß structure of amyloid fibrils, hence it is not possible to detect monomers or oligomers with the dye.111

Thioflavin T (ThT) is a fluorescent dye used to detect amyloid in tissue sections as well as in solutions of in vitro-formed amyloid. How ThT inter-acts with amyloid fibrils is not known, but it involves a 114 nm shift of the excitation maximum when ThT goes from an unbound (336 nm) to a bound state (450 nm), resulting in a fluorescence signal emitted at 482 nm.111,112 In this thesis, the dye was used to follow the aggregation process of amyloid-like fibrils in vitro in a 96 well-plate format.

The practical resolving power of modern electron microscopes is around 2 nm, i.e. hundred times greater than light microscopy. In transmission elec-tron microscopy amyloid is seen as straight unbranched fibrils with a diame-ter of 70-120 Å (Figure 4c). The individual protofilaments that are wound around each other may be visible in the micrographs.

Figure 4. Demonstration of amyloid with light and electron microscopy. A and B show amyloid stained with Congo red in normal and polarized light, respectively. C is an electron microscopical image of in vitro-formed amyloid-like fibrils of syn-thetic IAPP.

In the work of this thesis, the methods described above have been used for detection and visualization of amyloid in tissue as well as amyloid-like fibrils formed in vitro. However, many more methods exist. X-ray diffrac-tion has been utilized for many decades to study amyloid due to the specific pattern the fibrils give rise to. The pattern, which consists of a sharp reflec-tion of 4.7 Å and a more diffuse reflection at 10-11 Å, is attributed to the intersheet and intrasheet distances of the cross-ß structure.113 Atomic force microscopy and nuclear magnetic resonance are more recent techniques that

25

have been employed by the amyloid field and are mainly used to study the structure of the fibrils.14,15 In 2005, Nilsson et al introduced conjugated polyelectrolytes for detection of amyloid fibril formation and staining of amyloid in tissue.114,115 Crystal x-ray diffraction analysis has earlier been regarded as a method not suited for amyloid studies, as it has been very dif-ficult to produce crystals of amyloid fibrils. In the last years, however, suc-cessful attempts to grow nano- or microcrystal of small peptides with an amyloid-fibril structure have provided new facts on the molecular details of the fibril structure, including side-chain arrangements.17,18

Isolation and purification

In order to be able to study the nature and properties of amyloid proteins, purification of amyloid from affected tissues may be necessary. By repetitive homogenization of amyloid-containing tissue in 0.15 M NaCl followed by centrifugation, amyloid remains in the pellet material while the soluble pro-teins are found in the supernatant. Subsequent treatment of the pellet with acetone removes the fat portion of the tissue. Because of the stable structure, amyloid is very hard to dissolve. Guanidine-HCl, which is a chaotropic agent that breaks non-covalent bonds, is used for this purpose. After incubation in 6 M guanidine-HCl in 0.1 M Tris HCl and centrifugation, the amyloid fibril proteins are found enriched in the supernatant, separated from the non-dissolved cell debris and extracellular components in the pellet. Further puri-fication may be performed with gel filtration and reversed phase-high per-formance liquid chromatography (RP-HPLC), which separate proteins based on size and hydrophobicity, respectively.116

Characterization

As the therapies differ for the various amyloidoses it is of great importance to determine the identity of protein involved in the amyloid deposits. This is most commonly done with protein-specific antibodies in different applica-tions.

Immunohistochemistry

In immunohistochemistry antibodies are used to detect epitopes in tissue sections. Depending on the tissue and the antibody, different antigen re-trieval protocols can be employed. Antigenic retrieval is performed in order to make the epitopes more accessible to the antibody. For some amyloid proteins, the tissue sections need to be treated quite harshly, for example with formic acid. However, for the majority of the antibodies used in this thesis a mild treatment with preheated (90 °C) 0.02 M sodium citrate, pH 6, was used. The most commonly used immunohistochemistry protocol utilizes a system based on biotin/streptavidin combined with 3,3’-diaminobenzidine

26

tetrahydrochloride (DAB) for detection of antibody labeling. After incuba-tion with the primary antibody, a secondary antibody labeled with biotin is applied. In a third step, a conjugate consisting of streptavidin and horserad-ish peroxidase (HRP) is added. Streptavidin binds with a high affinity to biotin, thereby localizing HRP to the antibody complex that is bound to the protein of interest. The protein-antibody interaction is detected by the addi-tion of DAB. HRP oxidizes DAB to a brown color. Since the method is de-pendent on HRP, hydrogen peroxide is needed in the initial steps in order to extinguish the endogenous expression of peroxidases.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

and Western blot analysis

In SDS-PAGE proteins are separated according to size in a gel made from an acrylamide/bis-acrylamide matrix. Before separation, the proteins are mixed with sample buffer containing dithiothreitol, which reduces disulfide bridges, and SDS. SDS is an anionic detergent that denatures secondary structures and provides the proteins with a negative charge. The larger the protein the more negatively charged it will be. An electric field is applied and the proteins migrate towards the anode, according to their size. After separation, the proteins can be visualized by Coomassie blue staining, which stains all proteins in the gel. Since peptide polymers in the amyloid fibril are difficult to separate fully and the amyloid proteins often are truncated, amy-loid proteins separated by SDS-PAGE usually appear as a smear consisting of several bands representing monomers and polymers of the protein.

For protein identification, the proteins in the gel can be transferred to a membrane and then detected with an antibody-enzyme system (Western blot). In our laboratory we use a secondary antibody coupled to HRP and enhanced chemiluminiscence (ECL) for detection. HRP reacts with ECL, which emits light that is taken up by a film. After developing the film, the proteins are seen on the film as black bands.

Enzyme-linked immunosorbent assay (ELISA)

ELISA is a convenient detection method of antigens, commonly performed in a 96-well-plate format. The procedure varies between different laborato-ries depending on application. In the protocol of our laboratory, a protein solution is first coated to the wells. After blocking, a primary antibody di-rected against the protein of interest is added. A secondary antibody is then applied, to which alkaline phosphatase has been coupled. Alkaline phospha-tase converts a p-nitrophenyl phosphate substrate solution to a yellow color, which is detected at 405 nm.

27

Medin amyloid

The medial layer of the aorta is a very common site for amyloid infiltrates, an observation made by several studies.6,117-123 However, it was not until 1999 when the biochemical nature of this amyloid was unraveled. In the search for peripheral Aß deposition, aortic medial amyloid was investigated and a protein component of less than 6 kDa was purified. An eight amino acid residue peptide, with no resemblance to the AßPP sequence, was iso-lated from the component. A rabbit antiserum against the peptide was gener-ated and was shown to recognize medial amyloid specifically, implying that the peptide was derived from the amyloidogenic protein. The antiserum was further used in the purification and characterization of the amyloid from three aortas with extensive amyloid deposits. Extracted amyloid material was subjected to size-exclusion chromatography followed by RP-HPLC and SDS-PAGE. The separated proteins were transferred to a membrane, which was stained with Coomassie blue. The protein band of about 6 kDa, which also reacted with the antiserum in western blot analysis, was cut out and sequenced. Although somewhat ragged in the N-terminus, the main fibrillar protein was demonstrated to be composed of a 50 amino acid residue frag-ment. Due to its localization to the media this type of amyloid was named medin.5

Lactadherin - the precursor The sequence of the 6 kDa protein was compared with a sequence data base and was found to be identical to a peptide sequence in the C-terminal part of the lactadherin protein. With in situ hybridization it was further demon-strated that lactadherin is produced by the smooth muscle cells in the medial layer, a finding that was expected, given the local distribution of medin amy-loid.5

Most of the different amyloid proteins have been regarded as unrelated, with distinct amino acid sequences and native conformations. However, with improved sequence comparison tools and strategies it has become possible to trace evolutionary links between certain amyloid proteins. In one such study, lactadherin was found to share a common evolutionary origin with immuno-globulin light chains and transthyretin (involved in AL and TTR-related

28

amyloidoses, respectively), implicating that the amyloid proteins may be closer related than earlier anticipated.124

Structure

Lactadherin is 46 kDa large, consisting of 364 amino acid residues. The N-terminal part of the protein is composed of an epidermal growth factor (EGF)-like domain with an Arg-Gly-Asp (RGD) motif,125 which binds to �vß3 and �vß5 integrins on cells.126,127 The rest of the protein is divided into a C1- and C2-like domain, which have great homology to the C1 and C2 do-mains of coagulation factor V and VIII.125,128 The C2 domain interacts with anionic phospholipids, preferentially phosphatidylserine.127,129 Medin is lo-cated between amino acids 245-294 in the C2 domain (Figure 5).5 Lactad-herin is a glycoprotein with predominantly N-linked carbohydrate moieties, mainly in the second half of the protein. N-glycosidase digestion results in a molecular mass of about 41 kDa, which corresponds to the predicted mass of the polypeptide chain.130 It is a conserved protein with orthologues in mouse (MFG-E8), guinea pig (GP55), cow (PAS-6/7) and other animals.131 In addi-tion to the structure described above, lactadherin of these animals has an-other EGF-like domain N-terminally, but without the RGD motif.

Figure 5. Lactadherin consists of three major components; an EGF-like domain in the N-terminus is followed by a C1 and C2 domain. Medin is located in the C2 do-main.

How medin is formed from lactadherin is unclear, but the ragged N-terminal suggests that the fragment is generated by enzymatic cleavage of lactadherin rather than by alternative splicing.5 The crystal structure of the C2 domain of bovine lactadherin, in which medin is located, was only re-cently demonstrated.132,133 The domain consists mainly of ß-strands, ß-turns and random coil. Eight major ß-strands are packed into a distorted ß-barrel. Eleven shorter ß-strands, with only three amino acids each, come together at the top and bottom of the barrel, forming a flat upper surface and an uneven lower surface. Two important loop structures, spike 1 and 3, at the bottom of the barrel, contain solvent exposed hydrophobic residues. These amino acid residues form a hydrophobic patch, which presumably constitute the interac-tion interface between lactadherin and lipid moieties of cell membranes.133 Interestingly, spike 1 and 3 are positioned near the beginning and the end of the medin sequence. Enzymatic cleavage around the exposed hydrophobic patch would render a fragment similar to the determined medin sequence.

1 4744 202 207 245 294 364RGD

EGF-like domain C1 domain C2 domain

medinN C

29

Function

During the time course of the work with this thesis many reports describing lactadherin have been published. It has been identified in many different cells types, and various functions have been ascribed to the protein.

Phagocytosis

Lactadherin has been demonstrated to be involved in many phagocytic proc-esses of the body. In 2002, Hanayama et al discovered a factor that op-sonizes apoptotic cells for clearance by phagocytes. That factor was identi-fied as MFG-E8 or murine lactadherin. Apoptotic cells expose phosphatidyl-serine on their surfaces to which lactadherin binds with its C2 domain. Macrophages are drawn to the apoptotic cells by interaction via integrins to the RGD motif of lactadherin. Lactadherin thus functions as a bridge mole-cule that promotes engulfment of apoptotic cells.134 Mice with mutant MFG-E8 as well as MFG-E8 deficient mice displayed impaired phagocytosis of apoptotic cells, resulting in splenomegaly, glomerulonephritis and develop-ment of autoimmune disease due to elevated titers of autoantibodies.135,136

Furthermore, the lack of MFG-E8 has been correlated to accelerated athe-rosclerosis. Although both normal and atherosclerotic human arteries contain lactadherin, a lower expression was found in advanced atherosclerotic ves-sels. An atherosclerotic mice model deficient in MFG-E8 displayed more advanced plaques than MFG-E8+/+ mice, as a result of reduced phagocytosis of accumulated apoptotic cells and debris in the lesions.137

In addition of being the precursor of medin amyloid, lactadherin has also been coupled to another amyloidosis, namely Alzheimer’s disease. Lactad-herin is suggested to play a role in the clearance of Aß aggregates since macrophages from MFG-E8-/- mice showed decreased phagocytosis of the Aß peptide both in vitro and in vivo. Furthermore, reduced lactadherin levels were observed in brain tissue from patients with Alzheimer’s disease com-pared to age-matched controls. Particularly low levels of lactadherin were measured in areas rich in senile plaques. Recombinant human lactadherin was found to interact with Aß1-42 in a surface plasmon resonance assay, im-plicating that lactadherin targets the peptide and prepares it for phagocyto-sis.138

Carcinoma involvement

Lactadherin was first identified in milk and in sera from patients with breast cancer. The initial lactadherin research aimed at utilizing the protein as a diagnostic and therapeutic breast cancer marker. Sera from breast cancer patients contain lactadherin, in contrast to sera from healthy female controls, which have non-detectable levels.139 Passive immunization with a radiola-beled anti-lactadherin antibody has shown promising results in a study where

30

the antibody eradicated transplanted human breast tumors in a nude mouse model.140

An increased lactadherin expression is not restricted to breast cancer but has also been observed in other forms of carcinomas, such as ovarian, blad-der and colon carcinomas.141 The role of lactadherin there is not known. Given the interaction of lactadherin with integrins, Couto et al suggested the protein to affect cell polarity, position, growth and differentiation.125 Since then it has been shown that lactadherin may be involved in the malignant process by promoting vascular endothelial growth factor (VEGF)-dependent neovascularization. The association of VEGF to VEGF receptor 2 (VEGFR2) induces a chain of reactions, including engagement of tyrosine kinases, ultimately leading to endothelial cell survival, proliferation and migration. However, the VEGF-VEGF2R transduction pathway is dependent on a cross-talk with �vß3 and �vß5 integrins. Lactadherin was found to poten-tiate angiogenesis by interacting with the VEGF2R-associated integrins. Administration of VEGF to MFG-E8 deficient mice with a surgically in-duced ischemic hind limb did not stimulate neovessel formation. However, VEGF transfer to MFG-E8+/+ mice markedly improved vascularization.142 In a Rip-Tag2 mouse cancer model, where angiogenesis is critical, mice with MGF-E8 had more invasive tumors than MGF-E8-/- animals, which dis-played decreased tumor burden and reduced numbers of angiogenic islets.143

Protection against microbes

In milk lactadherin is found associated to anionic phospholipids of the milk fat globule membrane (MFGM). MFGM capsulates the fat of milk and is formed from the apical membrane of breast epithelial cells as milk is se-creted. Several studies demonstrate the capacity of milk lactadherin to pro-tect against rotaviral and Escherichia coli infections in breast-fed infants.144-

146 Newborns that are not breast-fed are more sensitive to rotaviral infec-tions, which give rise to gastroenteritis. Lactadherin prevents infection by binding to the virus, thereby blocking the virus from binding to the receptor on the host cell and preventing its entry into the cell.144 Because of the high degree of glycosylation, lactadherin is resistant to the gastric conditions of the newborn’s stomach, such as low pH and pepsin activity. It should be pointed out that the gastric pH value is higher and the pepsin activity lower in the newborn compared with the adult.147

Coagulation

Given the similarity of lactadherin and coagulation factor V and VIII, Shi and Gilbert studied lactadherin’s effect on coagulation. The results of their in

vitro study on bovine lactadherin indicate that lactadherin inhibits coagula-tion by competing with coagulation factor V and VIII for phospholipid-binding sites.148

31

Gonadogenesis and fertilization

The expression of lactadherin has been observed in early mouse go-nadogenesis and it has been found localized on the sperm plasma mem-brane.149-151 It binds to the zona pellucida of the unfertilized egg and plays a critical role during fertilization. MGF-E8 knockout mice showed reduced fertility and in vitro sperms from such mice were unable to bind to eggs. Data with different MFG-E8 recombinants suggest that interaction to the gamete surface is mediated by the C1 and C2 domains rather than the EGF-like domain.151

Exosomes

Exosomes are membrane nanovesicles secreted by many cells. Although their function remains obscure, the ability of the exosomes to interact with other cells indicates a communicative role. Exosomes from dendritic cells are enriched in antigen-presenting molecules and are therefore considered important in potentiating the immune response to pathogens and tumors.152 Lactadherin has been identified as a major component of dendritic exosomes and is thought to be involved in the docking of the phosphatidylserine-containing exosome to integrins on target cells.153,154 Other cell types have also been reported to secrete lactadherin-containing exosomes, such as fi-broblasts and adipocytes.155,156

Elastin interaction

In paper I of this thesis, we investigated the role of lactadherin in the vessel wall. Immunohistochemistry, for light and confocal microscopy, with poly-clonal anti-lactadherin antiserum showed that lactadherin coats the elastic fibers of human aortic material. In earlier work it was also demonstrated that medin amyloid appears in close association with elastic structures of arter-ies.6,157 Protein interactions were investigated in vitro using solid phase bind-ing assay and surface plasmon resonance (Biacore). With these methods lactadherin and medin showed a concentration-dependent interaction to tro-poelastin, the precursor of elastin. The data indicate that lactadherin binds to elastin via its medin domain, found in the C-terminal part of the protein. The physiological function of the lactadherin-elastin interaction may be of struc-tural significance. Since the EGF domain in the N-terminus has been shown to bind to integrins, it is possible that lactadherin in the aorta functions as a link that connects integrins on the smooth muscle cells to the elastic fibers. Further studies are needed to establish how medin and lactadherin bind to elastin. However, the highly hydrophobic nature of tropoelastin suggests that the proteins associate through hydrophobic interactions.

In addition to aortic tissue, we have also studied the distribution of lac-tadherin in normal and diseased dermis (unpublished results). An interaction to elastic fibers was observed also in skin. A particularly high lactadherin

32

immunostaining was seen associated to elastin in sun-damaged skin, such as basal cell and squamous cell carcinoma biopsies. In situ hybridization indi-cated that lactadherin is mainly produced by the epidermis. The observed expression of dermal lactadherin is partly in agreement of Watanabe et al who showed that mouse lactadherin is produced by epidermal keratinocytes of embryonal and newborn mice. Interestingly, they also noted an accumula-tion of MFG-E8 in sun-exposed skin from papillomas and carcinomas, al-though the protein was localized to the epidermis.158 In a recent study they further demonstrated that p63, a p53 family protein frequently over-expressed in squamous cell carcinomas, activates the transcription of MFG-E8, thereby providing an explanation for the strong MFG-E8 expression in carcinomas.159

Medin prevalence and distribution Medin amyloid (or senile aortic amyloid as it was called prior to its charac-terization) has mainly been studied in aging aortas, which shows the highest amyloid frequency.6,117-119,121,122 In one report from 1970, amyloid was found in all aortas from persons above 55 years of age.117 In a more recent investi-gation by Mucchiano et al, a similar prevalence was noted; medial amyloid was seen in 97% (61 of 63 cases) of the cases above 50 years, whereas only one out of 21 aortas below 50 years contained amyloid, clearly indicating that the prevalence of this type of amyloid increases with age.6 In yet another study 68 out of 100 aortas, from autopsied individuals over 60 years of age, contained amyloid.118 The difference in prevalence may be explained by the appearance of the majority of aggregates as minute nodules or thin streaks.6,119,122 Also, Congo red stains the aggregates rather weakly, render-ing the medial amyloid harder to detect compared to other amyloid types that commonly are larger and more birefringent. Different ethnicities may also contribute to the divergent prevalences.

The thoracic aorta contains more medin amyloid than the abdominal aorta.6,119,123 Data from one study on 18 Caucasian individuals, age 57-88 years, showed medin amyloid in 100% of the ascending thoracic aortas. Only 72% of the aortas contained amyloid in the abdominal part and the amount was significantly less: a total score of 24 as compared to 40 for the thoracic aorta.123 In a Japanese study on 224 autopsy cases over 40 years, medial amyloid was reported in 64% of the ascending aortic specimens and in 49% of the abdominal aortas.119

Other blood vessels and organs have been examined for the presence of medin amyloid. Mucchiano et al demonstrated that medial amyloid outside the aorta, in the temporal and carotid artery, exhibited a similar morphology as aortic amyloid and showed a positive reaction with an anti-serum devel-oped towards medial amyloid purified from aorta. It was therefore suggested

33

that the medial amyloid in the different vessels was of identical nature and part of a generalized vascular amyloid form.6 Later, when the biochemical nature of medin amyloid was determined, Peng et al utilized a medin-specific antiserum and mapped the distribution of medin amyloid throughout the vascular tree.123 Again, the highest prevalence of medin amyloid was found in the thoracic aorta, followed by the abdominal aorta. Outside the aorta deposits were most frequently observed in the basilar artery of the brain, where amyloid was found in 44% of the cases. The superior mesen-teric artery and the left coronary artery contained amyloid in 39% and 22%, respectively. Although only small amyloid spots were seen outside the aorta, the amyloid was usually found closely connected to the internal elastic lam-ina. The vein samples derived from the inferior vena cava were all nega-tive.123 The reason for this discrepancy is not known but may be related to the function of lactadherin as an elastin-binding protein and to the different elastin contents of the examined arteries. The aorta contains more elastin than the other arteries and thus presumably higher levels of lactadherin that may convert into amyloid.

Figure 6. A Congo-red stained section derived from the thoracic aorta (polarized light). Medin amyloid is deposited along the elastic fibers.

Medin amyloid occurs throughout the whole medial layer, but the depos-its are predominantly located to the inner half facing the intima6,120-122. The majority of the amyloid is found extracellularly and often in close proximity to the elastic fibers (Figure 6),6,117,157 although small aggregates within the smooth muscle cells have been observed.121,122 Medin amyloid has also in rare instances been observed in the intimal layer of the aorta and the coro-nary artery.5,123 However, it is important to point out that medin is not related to atherosclerosis,6,118-120,160 to which another amyloid type has been associ-ated (see below). On the contrary, Iwata and co-workers even saw less amy-loid in the media close to atherosclerotic lesions.119 The reason for this ob-servation is not discussed by the authors, but could possibly be explained by the finding by Ait-Oufella et al (see above), who demonstrated that human

34

atherosclerotic arteries contain less lactadherin than normal arteries.137 Re-duced amounts of the precursor protein would most likely result in less amy-loid. If these speculations hold true, there is a correlation between athero-sclerosis and medin amyloid, although inverse. Why lactadherin levels are lower during atherosclerosis is not known.

So far medin amyloid has only been observed in the wall of arteries and is regarded as a localized amyloidosis.3 The production of the fibril precursor close to the site of deposition and the identical deposition pattern of medin amyloid in different arteries suggest that the amyloidosis is a local form. However, given the occurrence of medin amyloid in arteries of many differ-ent organs one might argue that it is a systemic amyloidosis.

Medin and other vascular amyloid Medin amyloid is not the only amyloidosis affecting the vasculature. Arterial amyloid infiltrates are a general characteristic of almost all systemic amyloi-dosis, particularly AA, AL, and TTR amyloidoses. The reasons for the vas-cular depositions are unknown, but the infiltrates involve all three layers and may weaken the vessel walls with subsequent bleedings.

Localized amyloid is found associated to severe atherosclerotic lesions in the intima. Iwata et al showed that 27% of the severe lesions were positive for amyloid.119 In a later study, intimal amyloid was found in 35% of the atheromatous lesions in specimens above 50 years of age. The severity of the lesions was not mentioned, but no amyloid was detected in specimens below this age.6 There are still many unanswered questions regarding intimal amy-loid, including the biochemical nature of the protein as well as its involve-ment in the atherosclerotic process.

Intracranial vessels, mainly the leptomeningeal and cortical arteries of the cerebral lobes, are commonly affected by amyloid, a condition termed cere-bral amyloid angiopathy (CAA). CAA is in most cases formed by the Aß peptide, but other amyloid-related proteins may be involved, such as cys-tatin C,161 gelsolin,162 transthyretin163 and prion protein.164 Aß-derived CAA may arise in conjunction with Alzheimer’s disease or independently during aging. 80-90% of autopsied Alzheimer brains display CAA and 30% of the population above 60 years is estimated to be affected. CAA is classified as mild, moderate or severe. Mild CAA is defined by small amounts of amyloid deposits, restricted to the media around normal and atrophic smooth muscle cells. In moderate CAA the media has been replaced by amyloid and most of the smooth muscle cells have died. In severe CAA extensive amyloid depo-sition has accumulated resulting in a fragmented vascular wall with perivas-cular leakage of blood. It has been calculated that Aß-CAA is responsible for 15-20% of hemorrhagic strokes in the aging population. It is still debated from where the vascular Aß amyloid is derived. Both a local production in

35

the vessel wall as well as drainage of interstitial fluid from the brain have been proposed as potential sources of Aß.165

Due to the high frequency of different amyloid proteins in the aortic wall, we hypothesized that amyloid proteins of different biochemical nature may interplay and affect the deposition pattern of each other by a cross-seeding mechanism. In the last study of this thesis, paper IV, we examined the asso-ciation of AA amyloidosis and medin amyloid in the thoracic aorta. Four out of seven aortas displayed overlapping AA and medin aggregates when inves-tigated with double-labeling confocal microscopy. In thioflavin T experi-ments medin amyloid-like fibrils seeded human protein AA. However, fibrils of protein AA did not seed medin. The results clearly indicate that vascular amyloids may affect the distribution of each other. In this case, AA amyloid appears to be seeded and deposited by aggregates of medin. As medin amy-loid is found in almost all individuals above 50 years, medin may be one reason for the frequent deposition of systemic amyloidoses in the media of vessels.

Medin fibrillation Knowledge of how amyloid fibrils are formed is crucial in order to under-stand the molecular mechanisms of amyloidoses and for development of new treatments. The fibrillization of medin was examined in paper II of this the-sis. As expected, full-length medin formed amyloid-like fibrils in vitro. We also had access to shorter peptides within the medin sequence and aggrega-tion data from these were used to identify the regions essential for amyloid formation. Congo red-staining, thioflavin T fluorescence and transmission electron microscopy were methods used for fibril detection. Under the condi-tions tested the amyloid-promoting region was found localized to the 19 amino acid residues in the C-terminal part of medin. Furthermore, medin incubated with a SPOTs membrane, containing 21 decamer-peptides cover-ing the complete medin sequence with two amino acids overlap, interacted with the same C-terminal region, further demonstrating the importance of that region for medin-medin recognition and fibrillization. Earlier reports, from studies using the octapeptide NFGSVQFV (position 42-49 within medin), have proposed the two phenylalanines to be important for medin aggregation. Attractive interactions between the planar aromatic rings (�-stacking) have been suggested to contribute to the self-assembly of amyloid peptides into fibrils.166,167 However, in our study two peptides, with the phenylalanines substituted with alanines, still formed amyloid-like fibrils, thus implying that the aromatic residues are not essential for medin amyloid formation.

Given the extracellular location of the precursor lactadherin and the depo-sition of both lactadherin and medin amyloid along elastic structures, an

36

extracellular aggregation pathway, involving elastin as nucleation site, is the most likely (paper I). The importance of elastin for amyloid formation is further supported by the fact that medin amyloid has only been detected in arteries rich in elastin. However, at this point an intracellular fibrillation pathway of medin cannot be excluded. Intracellular medin amyloid is occa-sionally observed in arteries121,122 and medin aggregation has been reported to be accelerated in the presence of negative phospholipid membranes,168 which are solely localized in the inner half of the cell membrane. Intracellu-lar and extracellular aggregation may even occur in parallel and/or interact. It is possible that intracellular amyloid triggers and accelerates extracellular amyloid formation. Association of intracellular medin with negative phos-pholipid membranes would increase the local concentration of medin as well as alter the conformational state of the peptide, rendering it prone to aggre-gate into amyloid fibrils.168 During the intracellular fibrillization toxic oli-gomers may arise that could cause cell death. As the dead cell is resolved, the amyloid, which presumably is rather resistant to clearance, would remain and could function as a nucleus that seeds extracellular medin aggregation (Peng et al, unpublished results).

Consequences of medin deposition The existence of medin amyloid has been known for decades. However, the effects of medin amyloid have been very little studied, and only recently reports have emerged regarding the consequences of the deposition.

Thoracic aortic aneurysm and dissection

During the development of thoracic aortic aneurysm (TAA) and thoracic aortic dissection (TAD) the medial layer is weakened and could break, thus leading to a fatal bleeding. Although many risk factors, such as hyperten-sion, Marfan syndrome, smoking and atherosclerosis, have been associated with the diseases, the pathogenesis is in most cases poorly understood. Be-cause other forms of vascular amyloid, particularly CAA-affected arteries,165 show signs of medial degeneration and aneurysm formation, we believe that medin amyloid may have a similar effect in the aorta. Paper III shows the result of a study where the medin content in 27 TAA cases and 10 TAD cases was compared to 29 control specimens. The finding of significantly more medin amyloid in the control material was at first somewhat surprising. However, the medin immunolabeling was almost identical in the three groups, indicating that the two disease groups contain more medin im-munoreactive species that have not yet formed amyloid. Due to the increas-ing number of scientific reports describing noxious oligomeric assemblies (described earlier), one might speculate that the observed medin immunore-

37

activity may be such toxic species and could thus be an explanation for the degenerative changes in the media of TAA and TAD.

In a cell culture study we continued to examine the effect of medin on primary aortic smooth muscle cells. Fibrillar medin as well as newly dis-solved medin were non-toxic. However, non-fibrillar aggregates (Congo red-negative) of medin showed a moderate toxic activity. Furthermore, in a gela-tin zymography medin was observed to induce expression of different prote-ases, for example matrix metalloproteinase-2 (MMP-2). Upregulation of proteases have been observed in aneurysm specimens and these are believed to degrade the structural proteins of the vessel and contribute to medial de-generation.169-171 Interestingly, Aß also induces the expression of various proteases172,173 and elevated levels of MMPs have been observed in Alz-heimer brains.174,175 The results of paper III indicate that medin oligomers may be involved in the pathogenesis of thoracic aortic aneurysm and dissec-tion by weakening the aortic wall through induction of toxicity and matrix degrading enzymes.

Temporal arteritis

The occurrence of medin amyloid has been studied in the temporal artery, both with and without histological signs of giant cell arteritis (GCA).157 GCA is an inflammatory condition, with characteristic multinucleated giant cells as well as infiltrates of macrophages and T-lymphocytes. The inflam-mation may be severe and result in obstruction of the vascular lumen. The giant cells are often seen associated to the internal elastic lamina, which is commonly disrupted and fragmented during the inflammatory process. What causes temporal arteritis is not known, but an endogenous antigen, although unidentified, has been implicated to trigger the immune response.176,177 Amy-loid was, just like the components of immune response, found around the internal elastic lamina.157 A medin-specific antiserum labeled the amyloid, demonstrating that it was of medin origin. Fragmentation of the elastic lam-ina was a common finding. Interestingly, medin immunostaining of small elastin fragments was observed inside the giant cells. Although the non-inflamed arteries showed a higher prevalence (84%) than inflamed arteries (63%), it was concluded that medin amyloid may be of pathogenical impor-tance and induce an immune reaction in some of these individuals. The re-duced amyloid levels were explained by the clearance of medin amyloid by the giant cells.157 In this investigation medin immunolabeling was not quanti-fied. However, such a study would be of great interest. Given that non-amyloid medin aggregates appear to be involved in TAA and TAD, they may also be pathogenic in temporal arteritis.

Since the publication of the above study, many reports about the in-volvement of lactadherin in phagocytosis134-138 have appeared, as well as our work showing that lactadherin binds elastin (paper I). Although the results of

38

the study by Peng et al suggest medin as a potential antigen in GCA,157 it is tempting to speculate on lactadherin’s role in the inflammatory process of the disease. It is likely that the binding of lactadherin to the elastic lamina facilitates the interaction between elastin and the giant cells, thereby promot-ing the fragmentation of the elastic lamina. Peng et al further showed by in

situ hybridization that lactadherin was expressed evenly by the smooth mus-cle cells and the endothelial cells of non-inflamed arteries. However, in ar-teritis-affected vessels lactadherin expression was more uneven, clearly indi-cating that the expression is altered during the disease.157 The distribution of the lactadherin protein in inflamed and non-inflamed temporal arteries has not yet been investigated, but would likely provide further clues to lactad-herin’s role in GCA.

Artery stiffening

Several studies have reported on the close association of medin amyloid and the elastic structures of the arterial wall.6,117,157 In paper I we further demon-strated an interaction between medin and tropoelastin in vitro. The elasticity of the aorta depends mainly on three components: elastin, collagen and smooth muscle cells.178 Large artery stiffening is a hallmark of normal vas-cular ageing and results in increased blood pressure.179 It is likely that the deposition of medin amyloid on the elastic fibers contributes to this process by decreasing the flexibility. The observed toxicity of medin on smooth muscle cells (paper III) may reduce the elasticity even more. Also the prote-ase activity of the vessel wall has been linked to decreased elasticity. For example, the MMP-3 5A/5A genotype has been associated with stiffer larger arteries and higher systolic blood pressure, due to a four-fold increase in MMP-3 expression.180 The increased MMP-2 expression by medin (pa-per III) may have a similar effect, by degrading elastic structures of arteries.

39

Concluding remarks and future perspectives

In this thesis, which is based on four original papers, different aspects of medin amyloid have been studied, with the overall objective to investigate its fibrillation, the consequences of the deposits as well as the function of the precursor lactadherin.

In paper I we provide evidence that lactadherin interacts with elastin and we hypothesize that one normal function of lactadherin is to provide struc-ture to the vessel wall, by anchoring the elastic fibers to the smooth muscle cells. Medin amyloid is often found associated with the elastic fibers in situ and medin, just like lactadherin, showed a concentration-dependent binding to tropoelastin with surface plasmon resonance. The results indicate that the lactadherin-elastin interaction is mediated via the medin domain and that the elastic fiber may be central in medin amyloid development. As mentioned earlier, medin amyloid is only found in larger elastin-rich arteries. However, the effect of elastin on medin aggregation has not yet been investigated. Such studies have mainly been hampered by the insolubility of elastin, but the soluble precursor tropoelastin can be used in initial experiments. Incubat-ing soluble medin with amyloid-free aortic tissue sections may be another approach to study the role of elastin in medin aggregation. In such an ex-periment, formation of amyloid-like fibrils around elastin would strengthen our theory that the elastic fiber is critical for the fibrillation process.

How the medin fragment is cleaved and starts to aggregate is not known. However, in paper II we analyzed the fibrillation capacity of medin as well as of shorter peptides within the medin sequence and determined that the ability to form amyloid is attributed to the last 18-19 amino acid residues. Identification of the fibrillar core is of importance as a first step in the devel-opment of amyloid-inhibiting drugs.

In the second half of this thesis the involvement of medin amyloid in aor-tic pathology was examined. In paper IV, co-localization of medin and AA amyloid deposits in aortic tissue were observed and in vitro medin enhanced the formation of protein AA amyloid-like fibrils, indicative of a cross-seeding mechanism. These findings are interesting as they suggest that the frequent senile amyloid forms, found in many organs, may affect the fibrilli-zation and deposition of systemic amyloidoses, thereby potentiating the symptoms and progression of these lethal diseases.

During the last years it has become evident that protein aggregation is in-volved in many neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, transmissible spongiform encephalopathies and

40

amyotrophic lateral sclerosis. Although not all of these are amyloidoses, the protein aggregates are believed to be harmful and contribute to death of nerve cells by similar mechanisms. Paper III provides evidence that protein misfolding and aggregation are not solely restricted to diseases of the central nervous system but also are involved in disorders of the periphery. Aortas from patients suffering from thoracic aortic aneurysm and dissection (TAA and TAD, respectively) were found to contain less medin amyloid but more medin oligomers than control aortas. Furthermore, in cell culture medin stimulated the production of the elastase MMP-2 and non-fibrillar aggregates of medin induced toxicity. We therefore propose that medin oligomers con-tribute to weakened vessel wall and thus are involved in the pathogenesis of TAA and TAD, by reducing the smooth muscle cells and degrading the structural components of the aortic wall. Except for controlling associated cardiovascular risk factors, surgical repair of the degraded aortic wall is the only available treatment for TAA and TAD. For the future, drugs aimed to prevent medin formation and aggregation may be an appealing strategy to avoid development of TAA and TAD in some individuals. It is likely that toxic protein aggregates will be identified as pathogenic inducers of many more disorders, where the pathological mechanisms are still poorly under-stood.

Although the data presented in this thesis provide insights into several facets of medin and lactadherin, a wide range of issues remain unresolved and needs further examination. Issues of particular interest include: the effect of elastin in medin amyloid formation, the cleavage of lactadherin into medin, the involvement of medin amyloid in arterial stiffening and hyperten-sion, the role of lactadherin and medin in the inflammatory process of tem-poral arteritis and further characterization of the non-amyloid medin immu-nolabeling in aortas from TAA and TAD patients. An animal model would provide answers to many of these questions. In an attempt to find one, aortas of aging mice were stained for amyloid and medin. However, neither of these was observed (unpublished data). A transgenic mice model with lac-tadherin or medin over-expressed in the aortic wall may serve as a suitable animal model for such studies.

41

Populärvetenskaplig sammanfattning

Alzheimers sjukdom, galna-ko-sjukan och diabetes är sjukdomar som

har fått stor medial uppmärksamhet de senaste åren. Trots att symto-

men vid dessa sjukdomar skiljer sig mycket från varandra har de en

gemensam nämnare. Vid samtliga förekommer onedbrytbara protein-

klumpar, så kallade amyloid, som är skadliga för kroppen. Den vanli-

gaste amyloiden består av proteinet medin och finns i blodkärl hos näs-

tan alla personer över 50 år. Den här doktorsavhandlingen, som är

uppbyggd av fyra delarbeten, försöker klargöra hur denna amyloidtyp

byggs upp och om den kan leda till sjukdom.

Figur 1. En förenklad skiss över hur amyloid uppkommer.

Vår kropp består av tusentals olika proteiner. Dessa behövs för att bygga upp våra celler och vävnader samt ansvarar för majoriteten av de processer som dagligen äger rum i vår kropp. Ett protein byggs upp av drygt 20 olika typer av aminosyror. Det är våra gener som beskriver vilka aminosyror som ska finnas med och i vilken ordning de ska sitta. Strax efter att aminosyrorna kopplats ihop till en kedja veckas proteinet och det får sin korrekta tredimen-

Korrekt veckat protein (funktionellt)

Felveckat protein(ej funktionellt)

Amyloid - klump av felveckade protein

Oveckat protein -bestående av sammanlänkade aminosyror (ej funktionellt)Aminosyror

Oligomer - förstadium till amyloid (toxisk)

42

sionella struktur. Denna är nödvändig för proteinets funktion. Det finns sy-stem som kontrollerar att protein veckas rätt. Protein som missbildats bryts normalt ner, men ibland fungerar inte kontrollsystemen, vilket innebär att protein med en förändrad struktur inte tas om hand. Strukturförändringen kan då leda till att proteinet, eller delar av proteinet, klibbar ihop med andra felveckade proteinmolekyler och bildar stora, onedbrytbara klumpar, så kal-lade amyloid, i vävnaden (Figur 1).

Amyloid kan påvisas genom att färga den drabbade vävnaden med en färg som kallas Kongorött. I vanligt ljus ser amyloiden röd ut (Figur 2a) och i polariserat ljus blir den skimrande gul-grön (Figur 2b). Med elekronmikro-skopi ser man att amyloid består av långa trådlika strukturer, dessa benämns fibriller (Figur 2c).

Figur 2. Amyloid färgad med Kongorött (a-b). Amyloid byggs upp av protein som klumpat ihop sig till långa fibriller. Dessa är tydliga i elektronmikroskop (c).

I den amyloida formen har proteinet förlorat sin funktion. Dessutom är den skadlig för den drabbade vävnaden. Amyloid-ansamlingarna kan vara så stora att den normala vävnaden trängs bort. Organet fungerar då sämre eller i värsta fall inte alls. Vidare har man under det sista decenniet sett att för-stadier av amyloid, så kallade oligomerer (Figur 1), kan vara giftiga och därmed döda celler i omkringliggande vävnad. Vid Alzheimers sjukdom dödar amyloid-oligomerer nervcellerna och den drabbade personen blir de-ment.

Proteinet som bildar amyloid vid Alzheimers sjukdom kallas AßPP, men vid andra sjukdomar är andra proteiner inblandade. Vid t ex typ-2-diabetes är det IAPP som är det amyloida proteinet och vid Creutzfeldt-Jakobs sjuk-dom (den mänskliga formen av galna-ko-sjukan) bygger proteiner som kallas prioner upp amyloiden. Av de 28 kända amyloid-proteinerna är det inte alla som kopplats till sjukdom. Ett av dessa är lactadherin.

Lactadherin finns i många av kroppens vävnader. I stora kroppspulsådern eller aortan hittar man proteinet i muskellagret (Figur 3). I aortan finns även en mindre del av lactadherin, som kallas medin och det är denna del som

43

klumpar ihop sig och bildar amyloid (Figur 4). Medin-amyloid hittas i övre delen av aortan hos nästan alla människor över 50 år.

Figur 3. Ett blodkärl i genomskärning. En artär består av tre lager: intiman, median och adventitian. Det är i median eller muskellagret som medin-amyloid förekommer. Det är också detta lager som främst bidrar till kärlens elasticitet. Här finns nämligen de elastiska trådarna och de glatta muskelcellerna.

Figur 4. Medin-amyloid uppkommer genom att medin, som är en del av lactadherin, klyvs ut och börjar klumpa ihop sig till amyloid.

Lactadherins roll i aortan har tidigare varit okänd och i delarbete I under-söktes denna. Våra resultat visar att lactadherin fäster vid de elastiska trådar-na i aortan. Det är dessa trådar som tillsammans med de glatta muskelceller-na bidrar till kärlens elasticitet, vilken är viktig för att upprätthålla en funge-rande blodcirkulation. Det verkar vara medin-delen i lactadherin som är an-svarig för kopplingen till de elastiska trådarna. Det är ännu oklart vilken betydelse interaktionen har, men den skulle kunna bidra till kärlens upp-byggnad genom att lactadherin förankrar de elastiska trådarna med de när-liggande glatta muskelcellerna.

Hur medin-amyloid bildas är ännu oklart. I delarbete II tillverkades amy-loid i provrör av helt medin samt av olika delar av medin-proteinet, för att på så sätt komma fram till vilken del som är viktig för amyloid-uppkomsten. Resultat från studien visar att det går att bilda amyloid-lika fibriller av helt medin, som består av 50 aminosyror, men att det är de 18 sista aminosyrorna

medin

lactadherin

medin medin

medin

medinmedin

AMYLOID

adventitian

intiman

median = muskellagret blod

44

som är nödvändiga för den processen. Resultatet kan vara av vikt om man i framtiden vill framställa ett läkemedel mot medin.

Hittills har medin-amyloidens effekt i blodkärl varit okänd. På grund av alla amyloida proteiners liknande egenskaper misstänker vi att även medin har skadliga effekter på omkringliggande vävnad. I ett samarbete med thoraxkirurgen på Akademiska sjukhuset undersöktes om medin-amyloid kan leda till vidgning och därmed försvagning av kroppspulsådern, ett så kallat aorta-aneurysm. Ett aneurysm kan i värsta fall leda till att aortan spricker med en stor, livshotande blödning som följd. I delarbete III observe-rades att aneurysmpatienter inte har mer amyloid än kontrollpersoner med normalt dimensionerade aortor, men däremot mer medin-oligomerer. Vidare studerades medins eventuella giftighet genom att tillsätta medin-oligomerer till skålar innehållande glatta muskelceller. En toxisk effekt kunde då upp-mätas. Medin skulle därmed kunna leda till aneurysm genom att döda celler-na i kärlets muskellager och på så sätt försvaga kroppspulsådern.

Medin-amyloid skulle också kunna leda till förhöjt blodtryck, som bland annat orsakas av blodkärlens försämrade elasticitet. Data från delarbete I visar att medin, liksom lactadherin, interagerar med de elastiska trådarna och i ljusmikroskopi ser man tydligt att medin-amyloid ofta ligger dikt an mot de elastiska trådarna i aortan. Det är mycket troligt att elasticiteten påverkas negativt av amyloid-beläggningen och medin skulle därmed kunna ge upp-hov till högt blodtryck hos äldre.

Även andra amyloida proteiner påträffas i aortans muskellager och i delarbete IV undersöktes om medin-amyloid kan styra förekomsten av det amyloida proteinet SAA (serum amyloid A) till just aortan. SAA-relaterad amyloidos är en systemisk sjukdom, dvs den drabbar flera olika organ. Den uppkommer på grund av en kronisk inflammation, då man har förhöjda vär-den av SAA-proteinet som då kan börja klumpa ihop sig och bilda amyloid. Med mikroskop kunde vi se att i aortan hos patienter med SAA-relaterad amyloidos fanns två olika typer av ansamlingar, SAA- och medin-amyloid. I vissa fall kunde man till och med iaktta en blandning av dessa båda typer. Vidare observerades genom provrörsförsök att i närvaro av medin-amyloid bildades SAA-amyloid tidigare, än om medin-amyloid inte fanns närvaran-de. Resultaten visar således att medin har potential att påskynda amyloid-bildning och deponering av SAA till aortan. Fynden är av stor vikt eftersom de indikerar att en form av amyloid-inlagring kan sätta igång fibrill-bildningen av ett annat protein och därmed driva på utvecklingen av andra amyloid-sjukdomar.

Medin-amyloidforskningen är fortfarande i sin linda. Med den här av-handlingen börjar dock medin-amyloidens uppkomst och patologiska effek-ter att klargöras. Mer forskning behövs för att studera hur medin-delen skiljs från lactadherin, medins påverkan på blodtrycket och medin-oligomerernas roll vid aorta-aneurysm.

45

Acknowledgements

Arbetet i den här avhandlingen genomfördes på Institutionen för genetik och patologi vid Uppsala universitet med finansiellt stöd från Vetenskapsrådet, Hjärt- och Lungfonden och Europeiska Unionens sjätte ramprogram. De här åren på Rudbeck har innehållit mycket; spännande experiment, sti-mulerande diskussioner och läsning, men också långa arbetsdagar samt någ-ra portioner stress och frustration. Hur som helst så har det varit lärorika och roliga år. Jag har träffat många fantastiska människor som nu blivit nära vänner. Det finns många att tacka: Min handledare, Per Westermark - Tack för att du tog dig an mig som dokto-rand och för att du så generöst delat med dig av dina aldrig sinande amyloid-kunskaper. Tack också för din entusiasm över alla ”intressanta” (= oförklari-ga) resultat samt att jag fått arbeta självständigt och pröva mina egna vingar. Jag har lärt mig massor! Min bihandledare, Pär Gerwins - Tack för din hjälp med det molekylärbiolo-giska, för glada tillrop och dina visdomsord om livet som forskare. Mina medförfattare - Kul att få utbyta kunskaper och få samarbeta med er! Gunilla Westermark för goda råd och labbteknisk assistans. Ulf Hellman för sekvensering av synliga (och osynliga!) proteinband. Mina goa studenter, Mia, Lina, Linda O och Åsa, som hjälpt mig med de olika projekten. Mina gruppkollegor Anna Y, Elisabet, Eva, Jocke, Per, Siwei, Stina och Susanna - Med er var det aldrig tråkigt på labbet, resorna och fikarasterna. Tack för alla glada upptåg och för att ni alltid tar er tid att hjälpa till!! Mina kära skrivrumskollegor, Anna A, Carin, Helena B, Helena W, Linda P, Maria, Nurtena, Sara, och Stina, för all peppning och vänskap. Ni är fantas-tiska!

46

Gibbons Cornwell III - Thank you for reading and commenting this thesis! Tack också Johan, Helena P, Linda P, Sara och Stina för att ni tog er tid och läste delar av denna text och för lån av datorer. Amyloidgruppen i Linköping med Jana, Johan, Sebastian och Sofia. Tack för trevligt konferenssällskap, roliga fester och för att ni tog hand om mig när jag besökte ert labb! Alla andra glada kollegor i korridorerna som bidrar till den härliga stäm-ningen. Min kära familj och mina underbara vänner. Ni får jorden att snurra!

47

References

1. Virchow R: Zur Cellulose - Frage. Wirchows Arch Pathol Anat 1854, 6:416-426 2. Friedreich N, Kekulé A: Zur Amyloidfrage. Wirchows Arch Pathol Anat 1859,

16:50-65 3. Westermark P, Benson MD, Buxbaum JN, Cohen AS, Frangione B, Ikeda S,

Masters CL, Merlini G, Saraiva MJ, Sipe JD: A primer of amyloid nomencla-ture. Amyloid 2007, 14:179-183

4. Benson MD, James S, Scott K, Liepnieks JJ, Kluve-Beckerman B: Leukocyte chemotactic factor 2: A novel renal amyloid protein. Kidney Int 2008, 74:218-222

5. Häggqvist B, Näslund J, Sletten K, Westermark GT, Mucchiano G, Tjernberg LO, Nordstedt C, Engström U, Westermark P: Medin: an integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid. Proc Natl Acad Sci U S A 1999, 96:8669-8674

6. Mucchiano G, Cornwell GG, 3rd, Westermark P: Senile aortic amyloid. Evi-dence for two distinct forms of localized deposits. Am J Pathol 1992, 140:871-877

7. Dobson CM: Protein folding and misfolding. Nature 2003, 426:884-890 8. Dobson CM: Experimental investigation of protein folding and misfolding.

Methods 2004, 34:4-14 9. Barral JM, Broadley SA, Schaffar G, Hartl FU: Roles of molecular chaperones

in protein misfolding diseases. Semin Cell Dev Biol 2004, 15:17-29 10. Meusser B, Hirsch C, Jarosch E, Sommer T: ERAD: the long road to destruc-

tion. Nat Cell Biol 2005, 7:766-772 11. Yerbury JJ, Stewart EM, Wyatt AR, Wilson MR: Quality control of protein

folding in extracellular space. EMBO Rep 2005, 6:1131-1136 12. Yerbury JJ, Poon S, Meehan S, Thompson B, Kumita JR, Dobson CM, Wilson

MR: The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures. Faseb J 2007, 21:2312-2322

13. Wilson MR, Yerbury JJ, Poon S: Potential roles of abundant extracellular chap-erones in the control of amyloid formation and toxicity. Mol Biosyst 2008, 4:42-52

14. Harper JD, Lieber CM, Lansbury PT, Jr.: Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein. Chem Biol 1997, 4:951-959

15. Antzutkin ON, Leapman RD, Balbach JJ, Tycko R: Supramolecular structural constraints on Alzheimer's beta-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance. Biochemistry 2002, 41:15436-15450

16. Ippel JH, Olofsson A, Schleucher J, Lundgren E, Wijmenga SS: Probing solvent accessibility of amyloid fibrils by solution NMR spectroscopy. Proc Natl Acad Sci U S A 2002, 99:8648-8653

48

17. Nelson R, Sawaya MR, Balbirnie M, Madsen AO, Riekel C, Grothe R, Eisen-berg D: Structure of the cross-beta spine of amyloid-like fibrils. Nature 2005, 435:773-778

18. Makin OS, Atkins E, Sikorski P, Johansson J, Serpell LC: Molecular basis for amyloid fibril formation and stability. Proc Natl Acad Sci U S A 2005, 102:315-320

19. Makin OS, Serpell LC: Structures for amyloid fibrils. Febs J 2005, 272:5950-5961

20. Chiti F, Dobson CM: Protein misfolding, functional amyloid, and human dis-ease. Annu Rev Biochem 2006, 75:333-366

21. Fändrich M: On the structural definition of amyloid fibrils and other polypeptide aggregates. Cell Mol Life Sci 2007, 64:2066-2078

22. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL: Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 1998, 95:6448-6453

23. Janson J, Ashley RH, Harrison D, McIntyre S, Butler PC: The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles. Diabetes 1999, 48:491-498

24. Sousa MM, Cardoso I, Fernandes R, Guimaraes A, Saraiva MJ: Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy: evidence for toxicity of nonfibrillar aggregates. Am J Pathol 2001, 159:1993-2000

25. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M: Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002, 416:507-511

26. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ: Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 2002, 416:535-539

27. Reixach N, Deechongkit S, Jiang X, Kelly JW, Buxbaum JN: Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the ma-jor cytotoxic species in tissue culture. Proc Natl Acad Sci U S A 2004, 101:2817-2822

28. Nichols MR, Moss MA, Reed DK, Lin WL, Mukhopadhyay R, Hoh JH, Rosen-berry TL: Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy. Biochemistry 2002, 41:6115-6127

29. Necula M, Kayed R, Milton S, Glabe CG: Small molecule inhibitors of aggrega-tion indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem 2007, 282:10311-10324

30. Snow AD, Willmer J, Kisilevsky R: Sulfated glycosaminoglycans: a common constituent of all amyloids? Lab Invest 1987, 56:120-123

31. McCubbin WD, Kay CM, Narindrasorasak S, Kisilevsky R: Circular-dichroism studies on two murine serum amyloid A proteins. Biochem J 1988, 256:775-783

32. Ailles L, Kisilevsky R, Young ID: Induction of perlecan gene expression pre-cedes amyloid formation during experimental murine AA amyloidogenesis. Lab Invest 1993, 69:443-448

33. Ancsin JB, Kisilevsky R: The heparin/heparan sulfate-binding site on apo-serum amyloid A. Implications for the therapeutic intervention of amyloidosis. J Biol Chem 1999, 274:7172-7181

34. Li JP, Galvis ML, Gong F, Zhang X, Zcharia E, Metzger S, Vlodavsky I, Kis-ilevsky R, Lindahl U: In vivo fragmentation of heparan sulfate by heparanase

49

overexpression renders mice resistant to amyloid protein A amyloidosis. Proc Natl Acad Sci U S A 2005, 102:6473-6477

35. Castillo GM, Ngo C, Cummings J, Wight TN, Snow AD: Perlecan binds to the beta-amyloid proteins (A beta) of Alzheimer's disease, accelerates A beta fibril formation, and maintains A beta fibril stability. J Neurochem 1997, 69:2452-2465

36. Castillo GM, Cummings JA, Yang W, Judge ME, Sheardown MJ, Rimvall K, Hansen JB, Snow AD: Sulfate content and specific glycosaminoglycan back-bone of perlecan are critical for perlecan's enhancement of islet amyloid poly-peptide (amylin) fibril formation. Diabetes 1998, 47:612-620

37. Bergamaschini L, Rossi E, Storini C, Pizzimenti S, Distaso M, Perego C, De Luigi A, Vergani C, De Simoni MG: Peripheral treatment with enoxaparin, a low molecular weight heparin, reduces plaques and beta-amyloid accumulation in a mouse model of Alzheimer's disease. J Neurosci 2004, 24:4181-4186

38. Westermark GT, Norling B, Westermark P: Fibronectin and basement mem-brane components in renal amyloid deposits in patients with primary and secon-dary amyloidosis. Clin Exp Immunol 1991, 86:150-156

39. Lyon AW, Narindrasorasak S, Young ID, Anastassiades T, Couchman JR, McCarthy KJ, Kisilevsky R: Co-deposition of basement membrane components during the induction of murine splenic AA amyloid. Lab Invest 1991, 64:785-790

40. Merlini G, Bellotti V: Molecular mechanisms of amyloidosis. N Engl J Med 2003, 349:583-596

41. Pepys MB, Dyck RF, de Beer FC, Skinner M, Cohen AS: Binding of serum amyloid P-component (SAP) by amyloid fibrils. Clin Exp Immunol 1979, 38:284-293

42. Pepys MB, Booth DR, Hutchinson WL, Gallimore JR, Collins PM, Hohenester E: Amyloid P component. A critical review. Int J Exp Clin Invest 1997, 4:274-295

43. Tennent GA, Lovat LB, Pepys MB: Serum amyloid P component prevents pro-teolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis. Proc Natl Acad Sci U S A 1995, 92:4299-4303

44. Botto M, Hawkins PN, Bickerstaff MC, Herbert J, Bygrave AE, McBride A, Hutchinson WL, Tennent GA, Walport MJ, Pepys MB: Amyloid deposition is delayed in mice with targeted deletion of the serum amyloid P component gene. Nat Med 1997, 3:855-859

45. Hawkins PN, Myers MJ, Lavender JP, Pepys MB: Diagnostic radionuclide im-aging of amyloid: biological targeting by circulating human serum amyloid P component. Lancet 1988, 1:1413-1418

46. Wisniewski T, Frangione B: Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid. Neurosci Lett 1992, 135:235-238

47. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD: Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alz-heimer disease. Proc Natl Acad Sci U S A 1993, 90:1977-1981

48. Saunders AM, Strittmatter WJ, Schmechel D, George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BL, Gusella JF, Crapper-MacLachlan DR, Alberts MJ, et al.: Association of apolipoprotein E allele epsilon 4 with late-onset famil-ial and sporadic Alzheimer's disease. Neurology 1993, 43:1467-1472

49. Bales KR, Verina T, Cummins DJ, Du Y, Dodel RC, Saura J, Fishman CE, DeLong CA, Piccardo P, Petegnief V, Ghetti B, Paul SM: Apolipoprotein E is

50

essential for amyloid deposition in the APP(V717F) transgenic mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A 1999, 96:15233-15238

50. Stefani M, Dobson CM: Protein aggregation and aggregate toxicity: new in-sights into protein folding, misfolding diseases and biological evolution. J Mol Med 2003, 81:678-699

51. Lashuel HA, Lai Z, Kelly JW: Characterization of the transthyretin acid denatu-ration pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry 1998, 37:17851-17864

52. De Beer FC, Mallya RK, Fagan EA, Lanham JG, Hughes GR, Pepys MB: Se-rum amyloid-A protein concentration in inflammatory diseases and its relation-ship to the incidence of reactive systemic amyloidosis. Lancet 1982, 2:231-234

53. Esler WP, Wolfe MS: A portrait of Alzheimer secretases--new features and familiar faces. Science 2001, 293:1449-1454

54. Maury CP, Alli K, Baumann M: Finnish hereditary amyloidosis. Amino acid sequence homology between the amyloid fibril protein and human plasma gel-soline. FEBS Lett 1990, 260:85-87

55. Relini A, Canale C, De Stefano S, Rolandi R, Giorgetti S, Stoppini M, Rossi A, Fogolari F, Corazza A, Esposito G, Gliozzi A, Bellotti V: Collagen plays an ac-tive role in the aggregation of beta2-microglobulin under physiopathological conditions of dialysis-related amyloidosis. J Biol Chem 2006, 281:16521-16529

56. Kallberg Y, Gustafsson M, Persson B, Thyberg J, Johansson J: Prediction of amyloid fibril-forming proteins. J Biol Chem 2001, 276:12945-12950

57. Hsu AL, Murphy CT, Kenyon C: Regulation of aging and age-related disease by DAF-16 and heat-shock factor. Science 2003, 300:1142-1145

58. Chiti F, Calamai M, Taddei N, Stefani M, Ramponi G, Dobson CM: Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Proc Natl Acad Sci U S A 2002, 99 Suppl 4:16419-16426

59. Tjernberg L, Hosia W, Bark N, Thyberg J, Johansson J: Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides. J Biol Chem 2002, 277:43243-43246

60. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM: Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 2003, 424:805-808

61. Pedersen JS, Otzen DE: Amyloid-a state in many guises: survival of the fittest fibril fold. Protein Sci 2008, 17:2-10

62. Pedersen JS, Dikov D, Flink JL, Hjuler HA, Christiansen G, Otzen DE: The changing face of glucagon fibrillation: structural polymorphism and conforma-tional imprinting. J Mol Biol 2006, 355:501-523

63. Jarrett JT, Lansbury PT, Jr.: Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. Biochemistry 1992, 31:12345-12352

64. Jarrett JT, Lansbury PT, Jr.: Seeding "one-dimensional crystallization" of amy-loid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 1993, 73:1055-1058

65. Lundmark K, Westermark GT, Olsen A, Westermark P: Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism. Proc Natl Acad Sci U S A 2005, 102:6098-6102

66. Mabbott NA, MacPherson GG: Prions and their lethal journey to the brain. Nat Rev Microbiol 2006, 4:201-211

67. Lundmark K, Westermark GT, Nyström S, Murphy CL, Solomon A, Wester-mark P: Transmissibility of systemic amyloidosis by a prion-like mechanism. Proc Natl Acad Sci U S A 2002, 99:6979-6984

51

68. Johan K, Westermark G, Engstrom U, Gustavsson A, Hultman P, Westermark P: Acceleration of amyloid protein A amyloidosis by amyloid-like synthetic fi-brils. Proc Natl Acad Sci U S A 1998, 95:2558-2563

69. Solomon A, Richey T, Murphy CL, Weiss DT, Wall JS, Westermark GT, Westermark P: Amyloidogenic potential of foie gras. Proc Natl Acad Sci U S A 2007, 104:10998-11001

70. Falk E, Ladefoged C, Christensen HE: Amyloid deposits in calcified aortic valves. Acta Pathol Microbiol Scand [A] 1981, 89:23-26

71. Olsen KE, Westermark P: Amyloid in basal cell carcinoma and seborrheic kera-tosis. Acta Derm Venereol 1994, 74:273-275

72. Merlini G, Westermark P: The systemic amyloidoses: clearer understanding of the molecular mechanisms offers hope for more effective therapies. J Intern Med 2004, 255:159-178

73. Westermark P: Aspects on human amyloid forms and their fibril polypeptides. FEBS 2005, 272:5942-5949

74. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R: Physical basis of cognitive alterations in Alzheimer's disease: syn-apse loss is the major correlate of cognitive impairment. Ann Neurol 1991, 30:572-580

75. Lue LF, Kuo YM, Roher AE, Brachova L, Shen Y, Sue L, Beach T, Kurth JH, Rydel RE, Rogers J: Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease. Am J Pathol 1999, 155:853-862

76. Silveira JR, Raymond GJ, Hughson AG, Race RE, Sim VL, Hayes SF, Caughey B: The most infectious prion protein particles. Nature 2005, 437:257-261

77. Baglioni S, Casamenti F, Bucciantini M, Luheshi LM, Taddei N, Chiti F, Dob-son CM, Stefani M: Prefibrillar amyloid aggregates could be generic toxins in higher organisms. J Neurosci 2006, 26:8160-8167

78. Sirangelo I, Malmo C, Iannuzzi C, Mezzogiorno A, Bianco MR, Papa M, Irace G: Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F. J Biol Chem 2004, 279:13183-13189

79. Behl C, Davis JB, Lesley R, Schubert D: Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 1994, 77:817-827

80. Schubert D, Behl C, Lesley R, Brack A, Dargusch R, Sagara Y, Kimura H: Amyloid peptides are toxic via a common oxidative mechanism. Proc Natl Acad Sci U S A 1995, 92:1989-1993

81. Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Naga-shima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM: RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 1996, 382:685-691

82. Kawahara M, Kuroda Y, Arispe N, Rojas E: Alzheimer's beta-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium eleva-tions by a common mechanism in a hypothalamic GnRH neuronal cell line. J Biol Chem 2000, 275:14077-14083

83. Quist A, Doudevski I, Lin H, Azimova R, Ng D, Frangione B, Kagan B, Ghiso J, Lal R: Amyloid ion channels: a common structural link for protein-misfolding disease. Proc Natl Acad Sci U S A 2005, 102:10427-10432

84. Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, Johnson AB, Kress Y, Vinters HV, Tabaton M, Shimohama S, Cash AD, Siedlak SL, Harris PL, Jones PK, Petersen RB, Perry G, Smith MA: Mitochondrial abnor-malities in Alzheimer's disease. J Neurosci 2001, 21:3017-3023

52

85. Casley CS, Canevari L, Land JM, Clark JB, Sharpe MA: Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities. J Neurochem 2002, 80:91-100

86. Bucciantini M, Calloni G, Chiti F, Formigli L, Nosi D, Dobson CM, Stefani M: Prefibrillar amyloid protein aggregates share common features of cytotoxicity. J Biol Chem 2004, 279:31374-31382

87. Dobson CM: Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol 2004, 15:3-16

88. Douglas PM, Treusch S, Ren HY, Halfmann R, Duennwald ML, Lindquist S, Cyr DM: Chaperone-dependent amyloid assembly protects cells from prion tox-icity. Proc Natl Acad Sci U S A 2008, 105:7206-7211

89. Wechalekar AD, Hawkins PN, Gillmore JD: Perspectives in treatment of AL amyloidosis. Br J Haematol 2008, 140:365-377

90. Stangou AJ, Hawkins PN: Liver transplantation in transthyretin-related familial amyloid polyneuropathy. Curr Opin Neurol 2004, 17:615-620

91. Lachmann HJ, Goodman HJ, Gilbertson JA, Gallimore JR, Sabin CA, Gillmore JD, Hawkins PN: Natural history and outcome in systemic AA amyloidosis. N Engl J Med 2007, 356:2361-2371

92. Tagoe CE, Reixach N, Friske L, Mustra D, French D, Gallo G, Buxbaum JN: In vivo stabilization of mutant human transthyretin in transgenic mice. Amyloid 2007, 14:227-236

93. Sekijima Y, Dendle MA, Kelly JW: Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 2006, 13:236-249

94. Dember LM, Hawkins PN, Hazenberg BP, Gorevic PD, Merlini G, Butrimiene I, Livneh A, Lesnyak O, Puechal X, Lachmann HJ, Obici L, Balshaw R, Gar-ceau D, Hauck W, Skinner M: Eprodisate for the treatment of renal disease in AA amyloidosis. N Engl J Med 2007, 356:2349-2360

95. Barten DM, Albright CF: Therapeutic strategies for Alzheimer's disease. Mol Neurobiol 2008, 37:171-186

96. Hrncic R, Wall J, Wolfenbarger DA, Murphy CL, Schell M, Weiss DT, Solo-mon A: Antibody-mediated resolution of light chain-associated amyloid depos-its. Am J Pathol 2000, 157:1239-1246

97. Solomon A, Weiss DT, Wall JS: Therapeutic potential of chimeric amyloid-reactive monoclonal antibody 11-1F4. Clin Cancer Res 2003, 9:3831S-3838S

98. Solomon A, Weiss DT, Wall JS: Immunotherapy in systemic primary (AL) amyloidosis using amyloid-reactive monoclonal antibodies. Cancer Biother Ra-diopharm 2003, 18:853-860

99. Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG: Common structure of soluble amyloid oligomers implies common mecha-nism of pathogenesis. Science 2003, 300:486-489

100. Gilman S, Koller M, Black RS, Jenkins L, Griffith SG, Fox NC, Eisner L, Kirby L, Rovira MB, Forette F, Orgogozo JM: Clinical effects of Abeta immu-nization (AN1792) in patients with AD in an interrupted trial. Neurology 2005, 64:1553-1562

101. Pepys MB, Herbert J, Hutchinson WL, Tennent GA, Lachmann HJ, Gallimore JR, Lovat LB, Bartfai T, Alanine A, Hertel C, Hoffmann T, Jakob-Roetne R, Norcross RD, Kemp JA, Yamamura K, Suzuki M, Taylor GW, Murray S, Thompson D, Purvis A, Kolstoe S, Wood SP, Hawkins PN: Targeted pharma-cological depletion of serum amyloid P component for treatment of human amyloidosis. Nature 2002, 417:254-259

53

102. Cardoso I, Merlini G, Saraiva MJ: 4'-iodo-4'-deoxydoxorubicin and tetracy-clines disrupt transthyretin amyloid fibrils in vitro producing noncytotoxic spe-cies: screening for TTR fibril disrupters. Faseb J 2003, 17:803-809

103. Cardoso I, Saraiva MJ: Doxycycline disrupts transthyretin amyloid: evidence from studies in a FAP transgenic mice model. Faseb J 2006, 20:234-239

104. Chapman MR, Robinson LS, Pinkner JS, Roth R, Heuser J, Hammar M, Nor-mark S, Hultgren SJ: Role of Escherichia coli curli operons in directing amy-loid fiber formation. Science 2002, 295:851-855

105. Claessen D, Rink R, de Jong W, Siebring J, de Vreugd P, Boersma FG, Dijkhuizen L, Wosten HA: A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev 2003, 17:1714-1726

106. Fowler DM, Koulov AV, Alory-Jost C, Marks MS, Balch WE, Kelly JW: Functional amyloid formation within mammalian tissue. PLoS Biol 2006, 4:e6

107. Gazit E: Self-assembled peptide nanostructures: the design of molecular build-ing blocks and their technological utilization. Chem Soc Rev 2007, 36:1263-1269

108. Holmes TC, de Lacalle S, Su X, Liu G, Rich A, Zhang S: Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc Natl Acad Sci U S A 2000, 97:6728-6733

109. Scheibel T, Parthasarathy R, Sawicki G, Lin XM, Jaeger H, Lindquist SL: Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition. Proc Natl Acad Sci U S A 2003, 100:4527-4532

110. Bennhold H: Eine spezifische Amyloidfärbung mit Kongorot. Münchener Medizinische Wochenschrift 1922, 44:1537-1538

111. Westermark GT, Johnson KH, Westermark P: Staining methods for identifica-tion of amyloid in tissue. Methods Enzymol 1999, 309:3-25

112. Levine III H: Thioflavine T interaction with amyloid ß-sheet structures. Int J Exp Clin Invest 1995, 2:1-6

113. Eanes ED, Glenner GG: X-ray diffraction studies on amyloid filaments. J His-tochem Cytochem 1968, 16:673-677

114. Nilsson KP, Herland A, Hammarström P, Inganäs O: Conjugated polyelectro-lytes: conformation-sensitive optical probes for detection of amyloid fibril for-mation. Biochemistry 2005, 44:3718-3724

115. Nilsson KP, Hammarström P, Ahlgren F, Herland A, Schnell EA, Lindgren M, Westermark GT, Inganäs O: Conjugated polyelectrolytes--conformation-sensitive optical probes for staining and characterization of amyloid deposits. Chembiochem 2006, 7:1096-1104

116. Westermark GT, Westermark P: Purification of amyloid protein AA subspecies from amyloid-rich human tissues. Amyloid Proteins; Methods and Protocols. Edited by Sigurdsson EM. Totowa, New Jersey, Humana Press, 2005, pp 243-254

117. Schwartz P: Cardiovascular Amyloidosis. Amyloidosis, Cause and Manifesta-tion of Senile Detoriation. Springfield, Charles C Thomas, 1970, pp 80

118. Wright JR, Calkins E: Relationship of amyloid deposits in the human aorta to aortic atherosclerosis. A postmortem study of 100 individuals over 60 years of age. Lab Invest 1974, 30:767-773

119. Iwata T, Kamei T, Uchino F, Mimaya H, Yanagaki T, Etoh H: Pathological study on amyloidosis--relationship of amyloid deposits in the aorta to aging. Acta Pathol Jpn 1978, 28:193-203

120. Battaglia S, Trentini GP: [Aortic amyloidosis in adult life (author's transl)]. Virchows Arch A Pathol Anat Histol 1978, 378:153-159

54

121. Cornwell GG, 3rd, Westermark P, Murdoch W, Pitkanen P: Senile aortic amy-loid. A third distinctive type of age-related cardiovascular amyloid. Am J Pathol 1982, 108:135-139

122. Cornwell GG, 3rd, Murdoch WL, Kyle RA, Westermark P, Pitkänen P: Fre-quency and distribution of senile cardiovascular amyloid. A clinicopathologic correlation. Am J Med 1983, 75:618-623

123. Peng S, Glennert J, Westermark P: Medin-amyloid: A recently characterized age-associated arterial amyloid form affects mainly arteries in the upper part of the body. Amyloid 2005, 12:96-102

124. Stevens FJ: Possible evolutionary links between immunoglobulin light chains and other proteins involved in amyloidosis. Amyloid 2008, 15:96-107

125. Couto JR, Taylor MR, Godwin SG, Ceriani RL, Peterson JA: Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain. DNA Cell Biol 1996, 15:281-286

126. Taylor MR, Couto JR, Scallan CD, Ceriani RL, Peterson JA: Lactadherin (for-merly BA46), a membrane-associated glycoprotein expressed in human milk and breast carcinomas, promotes Arg-Gly-Asp (RGD)-dependent cell adhesion. DNA Cell Biol 1997, 16:861-869

127. Andersen MH, Graversen H, Fedosov SN, Petersen TE, Rasmussen JT: Func-tional analyses of two cellular binding domains of bovine lactadherin. Bio-chemistry 2000, 39:6200-6206

128. Larocca D, Peterson JA, Urrea R, Kuniyoshi J, Bistrain AM, Ceriani RL: A Mr 46,000 human milk fat globule protein that is highly expressed in human breast tumors contains factor VIII-like domains. Cancer Res 1991, 51:4994-4998

129. Andersen MH, Berglund L, Rasmussen JT, Petersen TE: Bovine PAS-6/7 binds alpha v beta 5 integrins and anionic phospholipids through two domains. Bio-chemistry 1997, 36:5441-5446

130. Cavaletto M, Giuffrida MG, Giunta C, Vellano C, Fabris C, Bertino E, Godo-vac-Zimmermann J, Conti A: Multiple forms of lactadherin (breast antigen BA46) and butyrophilin are secreted into human milk as major components of milk fat globule membrane. J Dairy Res 1999, 66:295-301

131. Mather IH, Banghart LR, Lane WS: The major fat-globule membrane proteins, bovine components 15/16 and guinea-pig GP 55, are homologous to MGF-E8, a murine glycoprotein containing epidermal growth factor-like and factor V/VIII-like sequences. Biochem Mol Biol Int 1993, 29:545-554

132. Lin L, Huai Q, Huang M, Furie B, Furie BC: Crystal structure of the bovine lactadherin C2 domain, a membrane binding motif, shows similarity to the C2 domains of factor V and factor VIII. J Mol Biol 2007, 371:717-724

133. Shao C, Novakovic VA, Head JF, Seaton BA, Gilbert GE: Crystal structure of lactadherin C2 domain at 1.7A resolution with mutational and computational analyses of its membrane-binding motif. J Biol Chem 2008, 283:7230-7241

134. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S: Identi-fication of a factor that links apoptotic cells to phagocytes. Nature 2002, 417:182-187

135. Hanayama R, Tanaka M, Miyasaka K, Aozasa K, Koike M, Uchiyama Y, Na-gata S: Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 2004, 304:1147-1150

136. Asano K, Miwa M, Miwa K, Hanayama R, Nagase H, Nagata S, Tanaka M: Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice. J Exp Med 2004, 200:459-467

55

137. Ait-Oufella H, Kinugawa K, Zoll J, Simon T, Boddaert J, Heeneman S, Blanc-Brude O, Barateau V, Potteaux S, Merval R, Esposito B, Teissier E, Daemen MJ, Leseche G, Boulanger C, Tedgui A, Mallat Z: Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice. Circula-tion 2007, 115:2168-2177

138. Boddaert J, Kinugawa K, Lambert JC, Boukhtouche F, Zoll J, Merval R, Blanc-Brude O, Mann D, Berr C, Vilar J, Garabedian B, Journiac N, Charue D, Sil-vestre JS, Duyckaerts C, Amouyel P, Mariani J, Tedgui A, Mallat Z: Evidence of a role for lactadherin in Alzheimer's disease. Am J Pathol 2007, 170:921-929

139. Ceriani RL, Sasaki M, Sussman H, Wara WM, Blank EW: Circulating human mammary epithelial antigens in breast cancer. Proc Natl Acad Sci U S A 1982, 79:5420-5424

140. Peterson JA, Couto JR, Taylor MR, Ceriani RL: Selection of tumor-specific epitopes on target antigens for radioimmunotherapy of breast cancer. Cancer Res 1995, 55:5847s-5851s

141. Carmon L, Bobilev-Priel I, Brenner B, Bobilev D, Paz A, Bar-Haim E, Tirosh B, Klein T, Fridkin M, Lemonnier F, Tzehoval E, Eisenbach L: Characteriza-tion of novel breast carcinoma-associated BA46-derived peptides in HLA-A2.1/D(b)-beta2m transgenic mice. J Clin Invest 2002, 110:453-462

142. Silvestre JS, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z: Lactadherin promotes VEGF-dependent neovascularization. Nat Med 2005, 11:499-506

143. Neutzner M, Lopez T, Feng X, Bergmann-Leitner ES, Leitner WW, Udey MC: MFG-E8/lactadherin promotes tumor growth in an angiogenesis-dependent transgenic mouse model of multistage carcinogenesis. Cancer Res 2007, 67:6777-6785

144. Yolken RH, Peterson JA, Vonderfecht SL, Fouts ET, Midthun K, Newburg DS: Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. J Clin Invest 1992, 90:1984-1991

145. Newburg DS, Peterson JA, Ruiz-Palacios GM, Matson DO, Morrow AL, Shults J, Guerrero ML, Chaturvedi P, Newburg SO, Scallan CD, Taylor MR, Ceriani RL, Pickering LK: Role of human-milk lactadherin in protection against symp-tomatic rotavirus infection. Lancet 1998, 351:1160-1164

146. Shahriar F, Ngeleka M, Gordon JR, Simko E: Identification by mass spectros-copy of F4ac-fimbrial-binding proteins in porcine milk and characterization of lactadherin as an inhibitor of F4ac-positive Escherichia coli attachment to intes-tinal villi in vitro. Dev Comp Immunol 2006, 30:723-734

147. Peterson JA, Hamosh M, Scallan CD, Ceriani RL, Henderson TR, Mehta NR, Armand M, Hamosh P: Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants. Pediatr Res 1998, 44:499-506

148. Shi J, Gilbert GE: Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites. Blood 2003, 101:2628-2636

149. Kanai Y, Kanai-Azuma M, Tajima Y, Birk OS, Hayashi Y, Sanai Y: Identifica-tion of a stromal cell type characterized by the secretion of a soluble integrin-binding protein, MFG-E8, in mouse early gonadogenesis. Mech Dev 2000, 96:223-227

150. Petrunkina AM, Lakamp A, Gentzel M, Ekhlasi-Hundrieser M, Topfer-Petersen E: Fate of lactadherin P47 during post-testicular maturation and ca-pacitation of boar spermatozoa. Reproduction 2003, 125:377-387

56

151. Ensslin MA, Shur BD: Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm-egg binding. Cell 2003, 114:405-417

152. Li XB, Zhang ZR, Schluesener HJ, Xu SQ: Role of exosomes in immune regu-lation. J Cell Mol Med 2006, 10:364-375

153. Morelli AE, Larregina AT, Shufesky WJ, Sullivan ML, Stolz DB, Papworth GD, Zahorchak AF, Logar AJ, Wang Z, Watkins SC, Falo LD, Jr., Thomson AW: Endocytosis, intracellular sorting, and processing of exosomes by den-dritic cells. Blood 2004, 104:3257-3266

154. Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 1999, 147:599-610

155. Ji H, Erfani N, Tauro BJ, Kapp EA, Zhu HJ, Moritz RL, Lim JW, Simpson RJ: Difference gel electrophoresis analysis of Ras-transformed fibroblast cell-derived exosomes. Electrophoresis 2008, 29:2660-2671

156. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N, Tamura T, Matsuda T: Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and hormone-dependent induction of milk fat glob-ule-epidermal growth factor 8-associated microvesicles. Endocrinology 2007, 148:3850-3862

157. Peng S, Westermark GT, Näslund J, Häggqvist B, Glennert J, Westermark P: Medin and medin-amyloid in ageing inflamed and non-inflamed temporal arter-ies. J Pathol 2002, 196:91-96

158. Watanabe T, Totsuka R, Miyatani S, Kurata S, Sato S, Katoh I, Kobayashi S, Ikawa Y: Production of the long and short forms of MFG-E8 by epidermal keratinocytes. Cell Tissue Res 2005, 321:185-193

159. Okuyama T, Kurata S, Tomimori Y, Fukunishi N, Sato S, Osada M, Tsukinoki K, Jin HF, Yamashita A, Ito M, Kobayashi S, Hata RI, Ikawa Y, Katoh I: p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/BA46 gene through interactions between the TA and DeltaN isoforms. Oncogene 2008, 27:308-317

160. Ishii T, Hosoda Y, Ikegami N, Shimada H: Senile amyloid deposition. J Pathol 1983, 139:1-22

161. Levy E, Lopez-Otin C, Ghiso J, Geltner D, Frangione B: Stroke in Icelandic patients with hereditary amyloid angiopathy is related to a mutation in the cys-tatin C gene, an inhibitor of cysteine proteases. J Exp Med 1989, 169:1771-1778

162. Kiuru S, Salonen O, Haltia M: Gelsolin-related spinal and cerebral amyloid angiopathy. Ann Neurol 1999, 45:305-311

163. Ushiyama M, Ikeda S, Yanagisawa N: Transthyretin-type cerebral amyloid angiopathy in type I familial amyloid polyneuropathy. Acta Neuropathol 1991, 81:524-528

164. Ghetti B, Piccardo P, Spillantini MG, Ichimiya Y, Porro M, Perini F, Kitamoto T, Tateishi J, Seiler C, Frangione B, Bugiani O, Giaccone G, Prelli F, Goedert M, Dlouhy SR, Tagliavini F: Vascular variant of prion protein cerebral amyloi-dosis with tau-positive neurofibrillary tangles: the phenotype of the stop codon 145 mutation in PRNP. Proc Natl Acad Sci U S A 1996, 93:744-748

165. Rensink AA, de Waal RM, Kremer B, Verbeek MM: Pathogenesis of cerebral amyloid angiopathy. Brain Res Rev 2003, 43:207-223

166. Reches M, Gazit E: Amyloidogenic hexapeptide fragment of medin: homology to functional islet amyloid polypeptide fragments. Amyloid 2004, 11:81-89

57

167. Gazit E: A possible role for pi-stacking in the self-assembly of amyloid fibrils. Faseb J 2002, 16:77-83

168. Olofsson A, Borowik T, Grobner G, Sauer-Eriksson AE: Negatively charged phospholipid membranes induce amyloid formation of medin via an alpha-helical intermediate. J Mol Biol 2007, 374:186-194

169. Jacob T, Ascher E, Hingorani A, Gunduz Y, Kallakuri S: Initial steps in the unifying theory of the pathogenesis of artery aneurysms. J Surg Res 2001, 101:37-43

170. Koullias GJ, Ravichandran P, Korkolis DP, Rimm DL, Elefteriades JA: In-creased tissue microarray matrix metalloproteinase expression favors proteoly-sis in thoracic aortic aneurysms and dissections. Ann Thorac Surg 2004, 78:2106-2110

171. Taketani T, Imai Y, Morota T, Maemura K, Morita H, Hayashi D, Yamazaki T, Nagai R, Takamoto S: Altered patterns of gene expression specific to thoracic aortic aneurysms: microarray analysis of surgically resected specimens. Int Heart J 2005, 46:265-277

172. Deb S, Wenjun Zhang J, Gottschall PE: Beta-amyloid induces the production of active, matrix-degrading proteases in cultured rat astrocytes. Brain Res 2003, 970:205-213

173. Jung SS, Zhang W, Van Nostrand WE: Pathogenic A beta induces the expres-sion and activation of matrix metalloproteinase-2 in human cerebrovascular smooth muscle cells. J Neurochem 2003, 85:1208-1215

174. Backstrom JR, Miller CA, Tokes ZA: Characterization of neutral proteinases from Alzheimer-affected and control brain specimens: identification of cal-cium-dependent metalloproteinases from the hippocampus. J Neurochem 1992, 58:983-992

175. Leake A, Morris CM, Whateley J: Brain matrix metalloproteinase 1 levels are elevated in Alzheimer's disease. Neurosci Lett 2000, 291:201-203

176. Weyand CM, Goronzy JJ: Medium- and large-vessel vasculitis. N Engl J Med 2003, 349:160-169

177. Weyand CM, Goronzy JJ: Giant cell arteritis as an antigen-driven disease. Rheum Dis Clin North Am 1995, 21:1027-1039

178. Wuyts FL, Vanhuyse VJ, Langewouters GJ, Decraemer WF, Raman ER, Buyle S: Elastic properties of human aortas in relation to age and atherosclerosis: a structural model. Phys Med Biol 1995, 40:1577-1597

179. Kingwell BA, Medley TL, Waddell TK, Cole TJ, Dart AM, Jennings GL: Large artery stiffness: structural and genetic aspects. Clin Exp Pharmacol Physiol 2001, 28:1040-1043

180. Medley TL, Kingwell BA, Gatzka CD, Pillay P, Cole TJ: Matrix metalloprote-inase-3 genotype contributes to age-related aortic stiffening through modulation of gene and protein expression. Circ Res 2003, 92:1254-1261

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 374

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-9275

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Medin amyloid - a matter close to the heart172508/FULLTEXT01.pdfList of papers This thesis is based...

Documents

Transcript of Medin amyloid - a matter close to the heart172508/FULLTEXT01.pdfList of papers This thesis is based...