TYPE XVIII AND XV COLLAGENS: PRIMARY STRUCTURE OF HUMAN ALPHA1(XVIII) CHAIN, PHENOTYPIC STUDIES OF TYPE XVIII COLLAGEN SINGLE NULL AND TYPE XVIII AND XV COLLAGEN DOUBLE NULL MICE

RITVAYLIKÄRPPÄ

Collagen Research Unit,Biocenter Oulu and

Department of Medical Biochemistryand Molecular Biology,

University of Oulu

OULU 2003

RITVA YLIKÄRPPÄ

TYPE XVIII AND XV COLLAGENS: PRIMARY STRUCTURE OF HUMAN ALPHA1(XVIII) CHAIN, PHENOTYPIC STUDIES OF TYPE XVIII COLLAGEN SINGLE NULL AND TYPE XVIII AND XV COLLAGEN DOUBLE NULL MICE

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in the Auditorium L101 of the Department ofMedical Biochemistry and Molecular Biology, on October24th, 2003, at 1 p.m.

OULUN YLIOPISTO, OULU 2003

Copyright © 2003University of Oulu, 2003

Supervised byProfessor Taina Pihlajaniemi

Reviewed byPh.D., B.Sc. Raymond Boot-HandfordProfessor Anders Kvanta

ISBN 951-42-7141-6 (URL: http://herkules.oulu.fi/isbn9514271416/)

ALSO AVAILABLE IN PRINTED FORMATActa Univ. Oul. D 753, 2003ISBN 951-42-7140-8ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESSOULU 2003

Ylikärppä, Ritva, Type XVIII and XV collagens: primary structure of humanalpha1(XVIII) chain, phenotypic studies of type XVIII collagen single null and typeXVIII and XV collagen double null mice Collagen Research Unit, Biocenter Oulu; Department of Medical Biochemistry and MolecularBiology, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland Oulu, Finland2003

Abstract

In this thesis study, the primary structure of the human α1(XVIII) polypeptide was elucidated, itstissue distribution was studied, and the phenotypic changes in the mouse eye due to lack of type XVIIIcollagen in a knock-out mouse model were studied further. In addition, the consequences ofsimultaneous lack of both type XVIII and XV collagen were studied in a mouse model lacking bothof these proteins.

Two variant forms of human α1(XVIII) polypeptide were identified in this study, although, todate, a third form has also been characterized. The analysis of tissue distribution of the twopolypeptide forms revealed differences in their tissue distribution, since the longest variant occursprominently in the liver, while the short form is the major transcript in other tissues studied, e.g. inthe kidney. The study of the type XVIII single null mouse eyes revealed abnormalities in the anterioreye segment in addition to the previously reported defects in the posterior eye part. In the type XVIIIsingle null mice the iris was fragmented, pigment deposits could be seen in the pupil, and the pupillaryruff in the edge of a normal mouse iris was missing in these mice. The ciliary body was also abnormal,since the ciliary processes start to show regression in adult animals and eventually the basal infoldingsof the non-pigmented ciliary body epithelia become flattened in the null mice. The intraocularpressure stabilizes to a lower level in adult mutant mice compared to controls, most likely reflectingthe atrophied ciliary epithelia. The BM zones were also defective in the type XVIII null mouse eyes.The absence of an immunosignal with one of the antibodies detecting laminin γ2 chain in the typeXVIII null mouse eyes may implicate conformational changes in the laminin γ2 chain due to lack oftype XVIII collagen, and subsequently interaction between type XVIII collagen and laminin γ2 chainin normal mouse eye BMs. The study of the type XVIII and XV double null mice revealed that thesemice were viable and fertile and had no major additional abnormalities compared to both single nullmice. However, the regression of hyaloid capillaries (vasa hyaloidea propria, VHP) was studied inthese mice, and a slight delay in the detachment of these vessels from the retina was noticed. Thus,the two collagens do not function entirely independently from each other.

The studies with type XVIII collagen single null mice indicate that in addition to the posterior eyephenotype, this collagen is needed for the normal structural integrity of the anterior eye segment andbasement membranes of the eye. The mouse model lacking both type XVIII and type XV collagenindicates that the roles of the two collagens are essentially diverse, although a slight compensatoryeffect was observed in the detachment of the hyaloid capillaries from the retina.

Keywords: anterior eye segment, basement membrane, eye, eye abnormalities, laminin,transgenic mice

AcknowledgementsThis study was carried out at the Department of Medical Biochemistry and MolecularBiology, University of Oulu, during the years 1994-2003.

I wish to express my deepest gratitude to my supervisor, Professor Taina Pihlajaniemi,for introducing me to the world of science and her guidance, patience and encouragementduring these years. I wish to express my sincere thanks to the Professor Kari Kivirikko forhis enthusiasm and creating excellent facilities for research work in the collagen field. Ialso wish to thank Professor Ilmo Hassinen, Professor Peppi Karppinen and DocentJohanna Myllyharju for their contribution in creating a scientific and inspiringatmosphere in the department.

I wish to thank Doctor Raymond Boot-Handford and Professor Anders Kvanta fortheir scientific expertise and valuable comments on the thesis. Anna Vuolteenaho is ack-nowledged for her careful revision of the language of the manuscript.

I wish to thank Docent Raija Sormunen for her expertise in ultrastuctural analysis andfor those numerous cheerful hours at the electron microscope and outside of it. I wish toacknowledge Professor Björn Olsen for his expertise in matrix biology and for his valu-able collaboration in this thesis. I wish to express my thanks to Doctor Naomi Fukai forhis invaluable collaboration. The co-authors of the papers presented in the thesis are ack-nowledged for their valuable contribution to the studies.

I wish to express my thanks to Marko Rehn for helping me to take my first steps in thefield of collagens and Janna Saarela for the teamwork in the human cDNA project, whichfelt endless at some point. I wish to thank Jaana Väisänen for introducing me the fascina-ting world of lab work and Maija Seppänen, Jaana Väisänen, Ritva Savilaakso, Eeva Leh-timäki, Sirpa Kellokumpu and Anna-Liisa Oikarainen for their excellent technical assis-tance. I wish to thank Pertti Vuokila for his help in many matters during these years. Iwish to thank Ari-Pekka Kvist, Juha Näpänkangas, Marko Kervinen and Risto Helminenfor helping me with the computers. Marja-Leena Kivelä, Auli Kinnunen, Seppo Lähdes-mäki and Marja-Leena Karjalainen are acknowledged for their kind help in numerouspractical matters. Seija Leskelä and Liisa Kärki are acknowledged for their precious helpin all the matters concerning posters and photographs.

I wish to express my warmest thanks to all the past and present people at the “happylounge” L120. Anu Muona, Timo Väisänen and Riikka Ylönen deserve my deepest gra-titude for their encouragement, support and sharing the ups and downs during these

countless years. I wish to express my thanks to Lauri Eklund for introducing me the worldof the mouse eye and the constructive and inspiring discussions during eye analysis. Iwish to thank all the people in the type XVIII group for their support and for creating agreat working atmosphere. I also want to express my warm thanks to Mirka Vuoristo andAnne Tuomisto for their friendship both in and out of work. I want to express my sincerethanks to Pirjo, Heli, Jaana and Sanna for their friendship over the past ten to nearly thirtyyears. I also want to acknowledge the “karonkka”-team for sharing the struggle with allthe practical matters concerning the thesis.

I owe my deepest gratitude to my sons Akseli and Kalle for their unquestioned loveand for their support in finding the ways to spend my leisure time. Heikki is also acknow-ledged for his help in matters of childcare. I wish to thank my parents Vappu and Toivofor their support during these years and my sister Ulla and brothers Esa and Ilpo and theirfamilies for their love and support in many aspects of life. I also wish to thank my auntsAsta and Kaisu for their support and warm care during these years.

This work was supported by grants from the Sigrid Juselius Foundation, the FinnishCultural Foundation, the Finnish Center of Excellence Programme (2000-2005) of theAcademy of Finland and the Support Foundation of the University of Oulu.

Oulu, September 2003 Ritva Ylikärppä

Abbreviationsaa amino acidsECM extracellular matrixBM basement membraneGly glycineX any amino acid (in Gly-X-Y triplet)Y any amino acid (in Gly-X-Y triplet)FACIT fibril-associated collagens with interrupted triple-helices

(and structurally related collagens)Multiplexin multiple triple-helix domains and interruptionsN- amino-C- carboxy-NC noncollagenousCOL collagenousα1(XV) α1 chain of type XV collagenα1(XVIII) α1 chain of type XVIII collagenkb kilobasesGAG glycosaminoglycan Col15a1-/- type XV collagen null alleleCol18a1-/- type XVIII collagen null alleleRT-PCR reverse transcription polymerase chain reactionIOP intraocular pressureVHP vasa hyaloidea propriaTVL tunica vasculosa lentis

List of original articlesThis thesis is based on the following articles, which are referred to in the text by theirRoman numerals:

I Saarela J, Ylikärppä R, Rehn M, Purmonen S & Pihlajaniemi T (1997) CompletePrimary Structure of Two Variant Forms of Human Type XVIII Collagen and Tis-sue-Specific Differencies in the Expression of the Corresponding Transcripts.Matrix Biol 16: 319–328.

II Ylikärppä R, Eklund L, Sormunen R, Kontiola AI, Utriainen A, Määttä M, Fukai N,Olsen BR & Pihlajaniemi T (2003) Lack of Type XVIII Collagen Results in AnteriorOcular Defects. FASEB J, in press.

III Ylikärppä R, Eklund L, Sormunen R, Muona A, Fukai N, Olsen BR & PihlajaniemiT (2003) Double knockout mice reveal a lack of major functional compensationbetween collagens XV and XVIII. Matrix Biol, in press.

IV Ylikärppä R, Eklund L, Sormunen R, Ilves M, Salo S, Niemelä M, Timpl R, OlsenBR & Pihlajaniemi T: Defective basement membranes in Col18a1 null mouse eyeand implications of interactions with laminin. Manuscript.

Contents

Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.1 The collagen family of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.2 Type XV collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.2.1 Genomic structures of human and mouse type XV collagen . . . . . . . . . 192.2.2 Polypeptide structure of type XV collagen . . . . . . . . . . . . . . . . . . . . . . 202.2.3 Tissue distribution of type XV collagen . . . . . . . . . . . . . . . . . . . . . . . . 212.2.4 Role of type XV collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.2.4.1 Type XV collagen-derived endostatin . . . . . . . . . . . . . . . . . . . 222.2.4.2 Mice lacking the type XV collagen present progressive

muscular dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.2.4.3 Type XV collagen in human diseases . . . . . . . . . . . . . . . . . . . 23

2.3 Type XVIII collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.3.1 Genomic structures of the human and mouse type XVIII collagen . . . . 232.3.2 Polypeptide structure of type XVIII collagen . . . . . . . . . . . . . . . . . . . . 242.3.3 Expression of type XVIII collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252.3.4 Role of type XVIII collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.3.4.1 Endostatin/NC1 domain of type XVIII collagen . . . . . . . . . . . 252.3.4.2 Receptors for type XVIII collagen-derived endostatin . . . . . . 262.3.4.3 Molecular interactions of type XVIII collagen . . . . . . . . . . . . 262.3.4.4 Human disease of defective type XVIII collagen . . . . . . . . . . 272.3.4.5 Mice lacking the type XVIII collagen . . . . . . . . . . . . . . . . . . . 27

2.4 The eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.4.1 The structure and development of the eye . . . . . . . . . . . . . . . . . . . . . . . 28

2.4.1.1 Development of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.4.1.2 Sclera-cornea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.4.1.3 Uvea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.4.1.4 Iris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.4.1.5 Ciliary body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.4.1.6 Retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.4.1.7 Cavities of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.4.1.8 Vasa hyaloidea propria (VHP) and tunica vasculosa

lentis (TVL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322.4.2 Does the mouse eye differ from the human eye? . . . . . . . . . . . . . . . . . . 332.4.3 Collagens of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342.4.4 Laminins of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2.4.4.1 Laminin-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.4.5 Anterior segment disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

2.4.5.1 Mouse models for anterior segment defects . . . . . . . . . . . . . . . 372.5 Basement membranes – active non-cellular entities of tissues . . . . . . . . . . . . . 37

3 Outlines of the present study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4.1 Characterization of the human α1(XVIII) cDNA clones (I) . . . . . . . . . . . . . . . 404.1.1 Isolation of the cDNA clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404.1.2 Sequence analysis of the cDNA clones . . . . . . . . . . . . . . . . . . . . . . . . . 414.1.3 Reverse transcription (RT) and polymerase chain reaction

(PCR) of human cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.2 Northern blot analysis of human α1(XVIII) transcripts (I) . . . . . . . . . . . . . . . 424.3 Type XVIII collagen null mice (II, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.4 Type XV and XVIII double null mice (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.5 Histological studies of mouse eyes (II-IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

4.5.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.6 Immunofluorescence studies of mouse eyes (II-IV) . . . . . . . . . . . . . . . . . . . . . 434.7 Ultrastructural analysis of mouse eyes (II, IV)) . . . . . . . . . . . . . . . . . . . . . . . . 434.8 Intraocular pressure measurement (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.9 Study of the function of the iris (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.10 Quantitative RT-PCR (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.11 Western blot analysis (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.12 Analysis of muscular defects (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454.13 Statistical analysis (II-IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465.1 The primary structure of human type XVIII collagen (I) . . . . . . . . . . . . . . . . . 465.2 Localization of the human α1(XVIII) transcripts (I) . . . . . . . . . . . . . . . . . . . . 475.3 Comparison of the human and mouse α1(XVIII) chains (I) . . . . . . . . . . . . . . . 475.4 Type XVIII null mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.4.1 Defects in anterior structures of the Col18a1-/- mouse eyes (II) . . . . . . 485.4.1.1 Iris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485.4.1.2 Ciliary body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

5.4.2 Ultrastructure of the BMs in Col18a1-/- mouse eyes (II, IV) . . . . . . . . . 495.4.3 BMs in Col18a1-/- iris and ciliary body (II) . . . . . . . . . . . . . . . . . . . . . . 49

5.4.3.1 Bruch’s and Bowman’s membrane (IV) . . . . . . . . . . . . . . . . . 495.4.4 Expression of basement membrane components in type XVIII

null and wild type mice (II, III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.4.4.1 Type XVIII collagen in mouse iris and ciliary body (II) . . . . . 50

5.4.4.2 Comparison of type XV and XVIII collagens in the mouse eye (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

5.4.4.3 Other BM components in the iris and the ciliary body (II) . . . 515.4.4.4 Expression of laminin-5 chains in the wild type

mouse eye (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515.4.4.5 Abnormal laminin staining pattern in Col18a1-/- mice (IV) . . 51

5.4.5 Western blot analysis (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525.4.6 Quantitative RT-PCR analysis of BMP-1 and MMP-2 expression (IV) 53

5.5 Comparison of Col15a1-/-; Col18a1-/- double null mice with single null mice (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535.5.1 No gross abnormalities in the double null mice . . . . . . . . . . . . . . . . . . 535.5.2 The regression of VHP and TVL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556.1 The type XVIII collagen polypeptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556.2 Extending the phenotype of Col18a1-/- mice . . . . . . . . . . . . . . . . . . . . . . . . . . 566.3 Study of the type XV and XVIII double null mice . . . . . . . . . . . . . . . . . . . . . . 58

7 Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60References

1 Introduction

Multicellular organisms, such as man and mouse, are composed of different tissues thatcontain various types of cells and extracellular matrix (ECM) produced by these cells.Collagens are proteins that are defined as structural components of ECM andcharacteristically have at least one triple-helical region containing polypeptides withrepeated Gly-X-Y amino acid triplets. To date, 27 different collagen types have beencharacterized and these collagens differ, in addition to their molecular structure, also intheir function, location, chain composition and in the amount of the protein in tissues.

The results presented in this thesis shed new light on one of the areas in the wide fieldof collagens and clarify the significance of one of the collagen subgroups, the multiple-xins, consisting of type XV and XVIII collagens. Type XV collagen has previously beensuggested to have a structural function in the skeletal muscle and capillaries, whereas typeXVIII collagen is required for the proper development of the eye vasculature. The workin this thesis provides information on the human type XVIII collagen polypeptide struc-ture and contributes to the phenotypic studies of type XVIII collagen null mice by furthercharacterizing the eye changes in these mice. Also some insights into the functionaldependency between the two collagens are provided with studies of mice lacking bothmembers of the multiplexin family.

2 Review of the literature

Tissues contain various types of cells and matrix (ECM) filling up the extracellular space.ECM is composed of heterogeneous proteins, glycoproteins, proteoglycans and othermacromolecules that form aggregates constituting a well-organized supramolecularnetwork. In addition to providing structural integrity and support for the cells, ECM alsoaffects the differentiation and proliferation as well as migration of the cells. Themetabolism of cells is also dependent on and regulated by ECM, since the matrixprovides a reservoir of molecules needed for numerous reactions in living cells. TheECM and cells are in constant interaction affecting the surrounding microenvironmentand having an impact also at the level of the whole tissue as well as the entire organ andorganism. The molecular composition of ECM varies depending on the spatial andtemporal state, but the most common molecules of ECM are the collagens, elastin andglycoproteins such as laminins and fibronectin. Basement membranes (BM) arespecialized structures of ECM existing throughout the body that underlie epithelial andendothelial cells and give physical support to cells and influence many biologicalprocesses. Collagens, especially the subfamily of multiplexins, BMs and laminin-5 of themolecular superfamily of laminins are looked at in more detail below, in addition to theeye, the organum visus, which is the main target of this study.

2.1 The collagen family of proteins

Proteins belonging to the superfamily of collagens have by definition at least one triple-helical region in their molecule and are structural components of the ECM. Collagens arecomposed of three α-chains that are either homo- or heterogeneous, and have Gly-X-Yrepeats in their structure enabling the formation of triple-helix. Due to its small size theglycine residue in the Gly-position fits into the center stabilizing the forming helix. The Xand Y positions of the triplets are frequently occupied by proline and hydroxyproline,respectively. Stability is increased by hydrogen bonds and water bridges within themolecule. The collagen molecules are further modified post-translationally both in andoutside the cell, i.e. many of the lysine residues besides proline are hydroxylated,

17

hydroxylysine residues can be glycosylated, intra- and interchain disulphide bonds areformed, proteins can be further glycosylated in the Golgi apparatus, glycosaminoglycanside chains may be added and proteolytic cleavage often occurs outside the cell, e.g.resulting in loss of procollagen propeptides in the fibrillar collagens. In addition tostructural support of the ECM, collagens also influence various biological activities of thecells. (van der Rest & Garrone 1991, Prockop & Kivirikko 1995, Myllyharju & Kivirikko2001.)

To date, altogether 27 different collagen types and over 40 genetically distinct α-chainshave been reported. Collagen types I–XIX are reviewed in several publications (van derRest & Garrone 1991, Bork 1992, Burgeson & Nimni 1992, Hulmes 1992, Kivirikko1993, Mayne & Brewton 1993, van der Rest & Bruckner 1993, Fichard et al. 1994, Fukaiet al. 1994, Brown & Timpl 1995, Prockop & Kivirikko 1995, Rehn & Pihlajaniemi1995a, Myllyharju & Kivirikko 2001), as well as genetic collagen diseases (Prockop &Kivirikko 1984, Prockop 1990, Kuivaniemi et al. 1991, Hudson et al. 1993, Kivirikko1993, Olsen 1995, Prockop & Kivirikko 1995, Sakai et al. 1996, Chan & Jacenko 1998,Myllyharju & Kivirikko 2001). The different collagen types, their tissue distribution andsubclassification are summarized in Table 1. Most of the collagens organize into supra-molecular aggregates and are divided into subgroups based on their structural or sequencesimilarities. The types I–III, V and XI form the subgroup of fibrillar collagens that haveuninterrupted Gly-X-Y repeats and form highly-ordered fibrils with 68 nm banding pat-tern in the extracellular space. Types XXIV and XXVII collagen appear to be new mem-bers of the fibrillar collagen family, although they have an interrupted triple helix and thesupramolecular structure is yet to be determined. The non-fibrillar collagens have inter-ruptions in their triple-helixes providing flexibility to the molecule and they are furtherdivided into several subgroups. The group of fibril-associated collagens with interruptedtriple-helixes (FACITs) are formed by the types IX, XII, XIV, XVI, XIX, XX and XXI,the transmembrane collagens are types XIII, XVII, XXIII and XXV, and the multiplexins(multiple triple-helix domains and interruptions) comprise the type XV and XVIII colla-gens that reside in basement membrane zones. Type IV collagen is also a basement mem-brane collagen, type VI collagen forms beaded filaments and type VII anchoring fibrils ofBM. Types VIII and X form hexagonal networks, whereas two of the newest collagentypes, namely XXII and XXVI, are yet unspecified as to their subgroup. (References forthe new collagens XX–XXVII are Koch et al. 2001, Fitzgerald & Bateman 2001, Chou &Li 2002, Tuckwell 2002, Banyard et al. 2003, Hashimoto et al. 2002, Sato et al. 2002,Boot-Handford et al. 2003, Koch et al. 2003, Pace et al. 2003. Complete mRNA sequenceon human α1(XXII) collagen is available in GenBank, accession number XM_291257.)

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Table 1. Collagen types, tissue distribution and subclassification.

The collagen subfamily of multiplexins has, to date, two members, type XV and XVIIIcollagen that have long N- and C-terminal noncollagenous (NC) regions with a collage-nous (triple-helical) domain interrupted by NC domains of varying length in between(Fig. 1). Both collagen types reside in the BM zones, although differences are found intheir tissue distribution. Type XV and XVIII collagen are proteolytically cleaved fromtheir C-terminus resulting in fragments with similar biological activities, since the clea-vage products are able to inhibit angiogenesis, as well as the migration of endothelialcells. However, there are also differences in the functional and binding properties of themultiplexin proteolytic fragments. The characteristics of multiplexin collagens are viewedin more detail below, starting with type XV collagen.

Type Tissue distribution Collagen subgroupI Most connective tissues Fibril-formingII Cartilage, vitreous humour, intervertebral disc Fibril-formingIII Most connective tissues Fibril-formingIV Basement membranes Nonfibril-formingV Tissues containing type I collagen Fibril-formingVI Most connective tissues Beaded filament-formingVII Many tissues, e.g. skin and cornea Anchoring fibril-formingVIII Many tissues, e.g. Descemet’s membrane Hexagonal lattice-formingIX Tissues containing type II collagen, e.g. cartilage and vitreous

bodyFACIT

X Hypertrophic cartilage Hexagonal lattice-formingXI Tissues containing type II collagen, e.g. cartilage and vitreous

bodyFibril-forming

XII Tissues containing type I collagen FACITXIII Many tissues, in low amounts TransmembraneXIV Tissues containing type I collagen FACITXV Basement membrane zones in many tissue MultiplexinXVI Many tissues FACITXVII Hemidesmosomes Anchoring filament-formingXVIII Basement membrane zones in many tissues MultiplexinXIX Basement membrane zones in many tissues FACITXX Minor components of several connective tissues FACITXXI Many tissues FACITXXII mRNA isolated from cartilage NDXXIII Cornea, lung, cartilage, amnion TransmembraneXXIV Bone, cornea Fibril-formingXXV Brain, neurons TransmembraneXXVI Testis, ovary NDXXVII Cartilage, eye, ear, lung, colon Fibril-formingData was collected from references appearing in the text. ND, not determined

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Fig. 1. The polypeptide domain structures of mouse type XV and XVIII collagens. Black regionscorrespond to the noncollagenous sequence and white to the collagenous ones. Putative N-terminal signal peptides are indicated by small, light grey boxes. The sequence specific to thelongest variant of mouse type XVIII collagen with ten cysteine residues (10C) is indicated by agrey box. Homologies to thrombospondin (Tsp) and Drosophila frizzled proteins (Fz) areindicated. C, cysteine residue. Numbers above the polypeptides indicate the potential GAGattachment site. The numbers in brackets under type XVIII collagen indicate the confirmedGAG attachment sites in the chicken type XVIII collagen (Dong et al. 2003).

2.2 Type XV collagen

2.2.1 Genomic structures of human and mouse type XV collagen

The gene encoding the α1 chain of human type XV collagen (COL15A1) and thecorresponding mouse gene (Col15a1) are located on the chromosomes 9q21-22 (Huebneret al. 1992) and 4B1-3 (Hägg PM et al. 1997a), respectively. Altogether 42 exons in thehuman gene encode the α1(XV) chain, the size of the gene being approximately 145kilobases (kb) (Hägg PM et al. 1998). The promoter region of the COL15A1 has severalSp1 binding sites, some of which appeared to be functional in HeLa cells (Hägg PM et al.1998), while the conventional TATA domain was lacking in the 5’ flanking region of thegene. The mouse gene for α1(XV) contains 40 exons and is about 110 kb in size, and thepromoter area of this gene lacks both TATA and CAAT boxes, but contains areas rich in Gand C (Eklund et al. 2000). The exon-intron organization was found to be conservedbetween the two species, although the region encoding the end of the N-terminal NCdomain and the beginning of the collagenous domain showed striking differences. The

C C

C C

10 C

C C C C C C

25 + 2aa

21 + 218aa

21 + 465aa299aa 674aa 315aa

XVIII

C C C C C C C C

579aa 507aa 265aa

25aaXV

NoncollagenousNC 1

CollagenousCOL 1-7 and NC 2-7

NoncollagenousNC 8

NoncollagenousNC 1

CollagenousCOL 1-10 and NC 2-10

NoncollagenousNC 11

Fz Tsp

Endostatin-likeregion

Endostatin

(1) (2) (3)

1 2

1 2 3 4

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mouse gene for type XV collagen lacked two of the exons present in the human geneencoding the N-terminal collagenous domain (Eklund et al. 2000). The 5’- flankingregions of the human and mouse genes for α1(XV) had four conserved domains, withcross-species sequence identities varying between 77% to 86% in the homologous areas(Eklund et al. 2000). Cis-acting elements for positive and negative regulation wereidentified in the functional analysis of the mouse promoter region of the gene coding forα1(XV), as studied in mouse NIH/3T3 cells (Eklund et al. 2000). Similar results werealso observed in promoter analysis of the human α1(XV) gene (Eklund et al. 2000).Moreover, cytokine regulation of type XV collagen mRNA levels has been described byKivirikko et al. (1999) using cultured human dermal fibroblasts and showing induction ofmRNA levels by TGF-β and subsequent reduction by TNF-α and IL-1β.

2.2.2 Polypeptide structure of type XV collagen

The first sequences of the primary structure of the α1 chain of type XV collagen werereported by Myers et al. (1992) and further completed by Kivirikko et al. (1994) andMuragaki et al. (1994). The primary structure of the human α1(XV) polypeptidecomprises 1388 residues with a 25-residue putative signal peptide, a 577-residuecollagenous domain flanked by a 530-residue N-terminal (NC1) and a 256-residue C-terminal (NC10) noncollagenous domains. The collagenous region contains ninecollagenous domains (COL1-9) varying between 15 and 114 residues in size, interruptedby eight noncollagenous domains (NC2-9) of 7 to 45 residues in size. An additional fiveshort imperfections of two to three residues in size in the Gly-X-Y sequence were foundin four of the collagenous domains (Kivirikko et al. 1994). Several putative sites for N-linked glycosylation and glycosaminoglycan (GAG) attachment have been found in thecentral collagenous sequence of the human α1(XV) polypeptide (Myers et al. 1992).Indeed, Li et al. (2000) reported that type XV collagen is a chondroitin sulfateproteoglycan in several human tissues. In their study they also found that the α chains oftype XV collagen are covalently linked by interchain disulfide bonds engaging two of theeight cysteine residues of the polypeptide. The N-terminal noncollagenous domain ofhuman type XV collagen contains an approx. 200-residue region with sequencehomology to the N-terminal domain of thrombospondin-1, a protein produced by theplatelets. This homology can also be found in many other collagens, namely types V, IX,XI, XII, XVI, XVIII, XXIV and XXVII (Bork 1992, Yoshioka et al. 1992, Pan et al.1992, Myers et al. 1993, Wälchli et al. 1993, Rehn and Pihlajaniemi 1994, Koch et al.2003, Boot-Handford et al. 2003). However, the amino acid residues involved in theheparin binding of thrombospondin are not conserved in the α1(XV) chain, leaving thesignificance of this homology unclear (Kivirikko et al. 1994).

The corresponding mouse polypeptide of type XV collagen (Fig. 1) has 1367 residueswith a putative 25-residue signal peptide, a 579-residue N-terminal noncollagenousdomain (NC1), a 507-residue collagenous sequence and a 256-residue C-terminal noncol-lagenous domain (NC8). The collagenous sequence of mouse α1(XV) polypeptide inclu-des seven collagenous domains (COL1-7) varying between 15 to 114 residues in size.These are separated by six noncollagenous regions (NC2-7) of 10 to 34 residues in size,and in addition, short imperfections of 2 or 3 residues in size can be found in four of the

21

seven collagenous domains of the mouse polypeptide (Hägg et al. 1997a). Nine putativesites for N-linked glycosylation and four putative sites for glycosaminoglycan attachmentwere found in the mouse α1(XV) polypeptide. The 200-residue region with homology tothe N-terminus of thrombospondin-1 is also found in the N-terminal noncollagenousdomain of mouse α1(XV) chain (Hägg et al. 1997a).

The human and mouse type XV collagen α1 polypeptide chains are homologous, sha-ring overall sequence identity of 72% and similarity of 91%. This homology is not, howe-ver, equally distributed along the polypeptide, but is most striking at the C-terminal endsof the α1(XV) chains (Hägg et al. 1997a). Both of the species have eight cysteine resi-dues in their α1(XV) chain in conserved positions in relation to each other. In addition,most of the putative glycosylations sites of α1(XV) are conserved between the species.The most striking difference between the human and mouse type XV collagen polypep-tide structure is the presence of two additional collagenous domains in the human chain.The additional domains in the human α1 (XV) chain are the 38-residue stretch includingthe first collagenous domain and the COL7 domain flanked by NC7 and NC8 domains,the area corresponding to the mouse NC6 domain (Hägg et al. 1997a).

2.2.3 Tissue distribution of type XV collagen

Type XV collagen resides in BM zones throughout the body. The distribution of type XVcollagen mRNAs in human tissues has been studied by Northern blot analysis and in situhybridization (Kivirikko et al. 1995a, Myers et al. 1996, Hägg et al. 1997b). Northernblot analysis of adult human tissues revealed strong expression of type XV collagenmRNA in heart, skeletal muscle, ovary, testis, small intestine, colon and placenta, andmoderate expression in the kidney, lung and prostate, and weak expression in thymus andpancreas (Kivirikko et al. 1995a, Myers et al. 1996). In contrast, type XV collagenmRNA was absent in the brain and liver. Human fetal lung and brain gave also positivehybridization patterns for type XV collagen mRNA, the latter signal being weak(Kivirikko et al. 1995a). The analysis of type XV collagen mRNA in mouse tissues byNorthern blot revealed strong hybridization signals in the heart and skeletal muscle,whereas the kidney, lung and testis showed moderate levels of α1(XV) chain transcript(Hägg et al. 1997b). By in situ hybridization the type XV collagen transcripts were foundto be synthesized by many cell types, especially mesenchymally derived cells,particularly muscle cells and fibroblasts. Also endothelial cells and some epithelial cellsin the kidney, lung, pancreas and placenta produce mRNA for type XV collagen(Kivirikko et al. 1995a).

The immunolocalizations of type XV collagen have shown that the protein exhibitswidespread tissue distribution and is located in BM zones, although some of the BMsremain negative for this collagen (Myers et al. 1996 and Hägg et al. 1997b, Myers et al.1997, Amenta et al. 2000, Muona et al. 2001, Tomono et al. 2002). Studies have beenmade to localize the human (Myers et al. 1996, Myers et al. 1997, Hägg et al. 1997b,Amenta et al. 2000 and Tomono et al. 2002) and mouse protein (Muona et al. 2001).Most of the capillaries show an immunosignal for type XV collagen, as does the BMzones of muscle cells (Hägg et al. 1997b). Also in the skin, the epidermal BM zone andthe BM of dermal capillaries, as well as the BM of large vessels are positive for type XV.

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In the lung, type XV collagen is located in BM of large vessels and airways (Myers et al.1996). However, the BM of fetal alveoli and some tubules in the developing kidney lacktype XV collagen (Hägg et al. 1997b). It is noteworthy that the type XV collagen immu-nosignal was not restricted solely to the BM zones, since some staining was observed alsoin the fibrillar collagen matrix (Myers et al. 1996). Ultrastructural analysis of the locationof type XV collagen indicated that type XV collagen resides in the outer region of laminadensa of the trophoblast BM, in the entire thickness of endothelial BM, and some signalwas also observed in the adjacent fibrillar matrix (Myers et al. 1996).

2.2.4 Role of type XV collagen

Some studies address the function of type XV collagen. A fragment of the C-terminus oftype XV collagen has been shown to have some functional importance. No humandisease caused by mutated or absent type XV collagen has been elucidated so far, butmice lacking type XV collagen have been generated revealing a potential structuralfunction for this collagen in muscle. A recent report suggested type XV collagen to be atumor suppressor gene based on mouse data (Harris 2003). The functional implications oftype XV collagen will be discussed in more detail in the following sections.

2.2.4.1 Type XV collagen-derived endostatin

The 185aa C-terminal fragment of type XV collagen corresponding to the region ofendostatin in type XVIII collagen is proteolytically cleaved, and the subsequent cleavageproduct, restin (related to endostatin), was first reported by Ramchandran et al. (1999).The homology of this fragment between human and mouse is significant throughout theentire 185aa region (72%), the identity being 60% (Ramchandran et al. 1999). The typeXV collagen-derived endostatin exhibits similar antiangiogenic and antimigratory effecton endothelial cells in vitro as the homologous endostatin domain of type XVIII collagen(Ramchandran et al. 1999, John et al. 1999, Sasaki et al. 2000). However, the type XVcollagen-derived fragment did not inhibit the proliferation of endothelial cells and themigration of non-endothelial cells (Ramchandran et al. 1999). Screening of humanhemofiltrate revealed circulating glycosylated C-terminal fragments of type XV collagen(John et al. 1999).

2.2.4.2 Mice lacking the type XV collagen present progressive muscular dystrophy

An approach to functional analysis of type XV collagen was made by generating micebearing a mutated type XV collagen gene. A null mutation in the mouse Col15a1 genewas introduced by gene targeting (Eklund et al. 2001). The absence of type XV collagen

23

in the mice did not have any major effect on their development, reproduction and vitality.However, after 3 months of age they showed histological changes characteristic ofmuscular diseases, and these mice were more susceptible to muscular injuries afterexercise. Also cardiac injuries resembling those in early or mild heart disease werepresent after exercise and perfused hearts exhibited a diminished inotropic response.Although the restin fragment of type XV collagen has an antiangiogenic role, the vasculardevelopment was shown to be normal in the Col15a1-/- mice, while ultrastructuralanalysis revealed abnormal capillaries and endothelial cell degeneration in the heart andthe skeletal muscle of these mice. (Eklund et al. 2001). This study suggests that type XVcollagen has a structural role in skeletal muscle and capillaries by stabilizing cells withthe surrounding connective tissue, as discussed by Eklund et al. (2001).

2.2.4.3 Type XV collagen in human diseases

Defective type XV collagen or its abnormal expression has not been found to cause anyhuman disease so far, but abnormally elevated expression of type XV collagen has beendescribed in the renal interstitial matrix in patients with kidney fibrosis due to differentpathological processes (Hägg et al. 1997b). The study of Amenta et al. (2000) describestype XV collagen in colonic adenocarcinomas and shows that the distribution of type XVcollagen differs from the other BM proteins, namely type IV collagen and laminin, bybeing absent in BM zones of malignant glandular elements in moderately differentiatedtumors. There was no type XV collagen in the malignant tumor epithelium BM zones,either, but the interstitial tumor stroma contained high levels of this collagen. Thepossible role of type XV collagen in tumor progression and invasion remains to beelucidated.

2.3 Type XVIII collagen

2.3.1 Genomic structures of the human and mouse type XVIII collagen

The genes coding for the α1 chain of type XVIII collagen have been mapped to humanchromosome 21q22.3 and to mouse chromosome 10 (Oh et al. 1994a). The completeexon-intron organization of the human COL18A1 gene revealed altogether 43 exons, thesize of the gene being approx. 105 kb (Suzuki et al. 2002, Elamaa et al. 2003). 43 exonshave also been found for the mouse Col18a1 gene of over 102 kb in size, and it is highlysimilar in structure to the human COL18A1 gene (Rehn et al. 1996). The three forms oftype XVIII collagen are derived from transcripts resulting from the use of two promoters,at a distance of 50 kb (Rehn et al. 1996). Promoter 1 directs the synthesis of the shortestform, the NC1-301 in the mouse, the exons 1 and 2 specifically encoding this form.Promoter 1 lacks the conventional TATAA and CCAAT motifs, while it contains three GC

24

boxes typical of housekeeping genes (Rehn et al. 1996). Promoter 2 directs the synthesisof the two longest forms starting from exon 3 specifically encoding the NC1-517 and theNC1-764 variants in mouse, and it lacks the TATAA and GC boxes, but contains CCAATand CTC motifs (Rehn et al. 1996, Lietard et al. 2000). Furthermore, exon 3 undergoesalternative splicing resulting in the two N-terminal sequences differing in length. Theexpression of the longest forms of type XVIII collagen is controlled through ubiquitous(Sp1) and liver-specific (HNF3/NF1) regulatory elements (Lietard et al. 2000).

2.3.2 Polypeptide structure of type XVIII collagen

The primary structure of the type XVIII collagen α1 chain was first elucidated nearly tenyears ago (Oh et al. 1994a and b, Rehn and Pihlajaniemi, 1994, Rehn et al. 1994) andsubsequently it revealed three forms of the polypeptide in mouse (Fig. 1, Muragaki et al.1995, Rehn and Pihlajaniemi 1995b). The partial primary structure of human α1(XVIII)was reported by Oh et al. (1994a). The first characterization of the complete primarystructure of the human α1 (XVIII) is included in this thesis, and also supplementedelsewhere (Suzuki et al. 2002). The polypeptides of mouse type XVIII collagen vary intheir N-terminal sequences, but they all share a 299-residue portion of the NC1 domain, a674-residue collagenous domain and a 315-residue NC11 domain at their C-terminus.The shortest form, the 1315-residue α1(XVIII) chain contains a 25-residue putative signalpeptide and a 301-residue NC1 region, while the two longest forms have a different 21-residue signal sequence and NC1 regions of 517 and 764 residues in polypeptides of 1527and 1774 residues in length, respectively (Muragaki et al. 1995, Rehn and Pihlajaniemi1995b). The longest form of type XVIII collagen is characterized by a 110-residuesequence with 10 cysteine residues in the NC1 domain. This sequence is homologous tothe frizzled proteins belonging to the family of G-protein-coupled membrane receptors,but the significance of this homology remains to be further elucidated. (Rehn andPihlajaniemi, 1995b). The 1527-residue polypeptide form results from alternative splicingof the longest polypeptide transcript resulting in loss of 247 aa residues containing thefrizzled motif (Rehn and Pihlajaniemi, 1995b). The central region of the α1(XVIII)chains has 10 collagenous domains (COL1-10) of 18 to 122 residues interrupted byseveral NC regions (NC2-10) of 10 to 24 residues (Oh et al. 1994b, Rehn et al. 1994,Rehn and Pihlajaniemi, 1995b).

Type XVIII collagen has several putative glycosylation sites in its primary structure(Oh et al. 1994b, Rehn and Pihlajaniemi, 1994, Rehn and Pihlajaniemi, 1995b) andindeed, Halfter et al. (1998) showed that type XVIII collagen is a heparan sulfate proteog-lycan. Dong et al. (2003) studied the glycosylation of recombinant chick type XVIII col-lagen and detected three GAG side chains in the middle and N-terminal part of the coreprotein. A part of the N-terminal NC1 domain common to all variants shares homology tothe N-terminal domain of thrombospondin, but since the aa residues involved in the hepa-rin binding of thrombospondin are not conserved in type XVIII collagen, the significanceof this homology remains unclear (Rehn and Pihlajaniemi, 1994).

25

2.3.3 Expression of type XVIII collagen

In addition to man and mouse, the type XVIII collagen has also been characterized inchicken, C.elegans and frog (Halfter et al. 1998, Ackley et al. 2001, Elamaa et al. 2002,Dong et al. 2003). The tissue distribution of type XVIII collagen in in these species hasbeen studied by Northern blot analysis, in situ hybridization and by immunologicalstudies both at light and electron microscopy level (Oh et al. 1994a, b, Rehn andPihlajaniemi 1994, Rehn and Pihlajaniemi 1995b, Muragaki et al. 1995, Saarela et al.1998, Halfter et al. 1998, Musso et al. 1998, Sasaki et al. 1998, Miosge et al. 1999,Inoue-Murayama et al. 2000, Sasaki et al. 2000, Ackley et al. 2001, Musso et al. 2001,Lin HC et al. 2001, Elamaa et al. 2002, Fukai et al. 2002, Tomono et al. 2002, Kato et al.2003). Type XVIII collagen is localized to BM zones of a variety of embryonic and adultmouse and human tissues, particularly those associated with blood vessels, BM zone ofskin epidermis, Bowman’s capsule as well as tubules and arterioles in the kidney, choroidplexus of the brain and the sinusoidal and portal BM zones of the liver (Muragaki et al.1995, Saarela et al. 1998). The expression of this collagen in the eye has also beenstudied (Halfter et al. 2000, Lin HC et al. 2001, Fukai et al. 2002, Kato et al. 2003). Thethree variants of this collagen are expressed in a tissue-specific manner in BM zonesthroughout the body, the shortest form localizing especially to BM zones of blood vesselendothelia and the long spliced form of type XVIII collagen to the liver BMs (Muragakiet al. 1995, Rehn and Pihlajaniemi 1995b, Saarela et al. 1998).

2.3.4 Role of type XVIII collagen

The function of type XVIII collagen is still unclear, although the C-terminal fragment ofthis collagen, endostatin, has been shown to have functional significance in endothelialcell migration and proliferation and thus, to be potentially involved in antiangiogenicprocesses and in morphological events (O’Reilly et al. 1997, Lin Y et al. 2001, Ortega &Werb, 2002, Vainio et al. 2003). Numerous studies on the properties and mechanism ofaction of endostatin have been reported, but it is still not clear which findings are ofphysiological significance. It is likely that the role of this molecule is not limited to theactivity of its C-terminal fragment; it is probable that also other parts of the molecule,namely the N-terminal NC domain and collagenous domain, have functional propertiesthat still need to be unraveled. In this section, a set of studies implicating the function oftype XVIII collagen is discussed.

2.3.4.1 Endostatin/NC1 domain of type XVIII collagen

In recent years, the C-terminal part of the type XVIII collagen molecule has gained a lotof attention due to its antiangiogenic activity and its potential to regress tumor growthand to suppress tumor-induced angiogenesis (O’Reilly et al. 1997, Boehm et al. 1997,Dhanabal et al. 1999a). There are several reports both for and against the antitumor effect

26

of endostatin (O’Reilly et al. 1997, Boehm et al. 1997, Dhanabal et al. 1999a, Jouanneauet al. 2001, Huang et al. 2001, Kisker et al. 2001, Eisterer et al. 2002, Pawliuk et al.2002, Peroulis et al. 2002, Ye et al. 2002, Dkhissi et al. 2003, Nakashima et al. 2003).The endostatin fragment of type XVIII collagen can be generated by cathepsin-L, elastaseor by matrilysin cleavage from the parental molecule (Wen et al. 1999, Felbor et al. 2000,Lin HC et al. 2001). Studies with recombinant NC1 fragment of type XVIII collagenhave demonstrated that this domain consists of an N-terminal association region (approx.50 residues), a central, protease-sensitive hinge region (approx. 70 residues) and a C-terminal endostatin domain of approx. 180 residues in size and (Sasaki et al. 1998). TheNC1 domain of type XVIII collagen is able to induce motility of both endothelial andnon-endothelial cells, while the endostatin fragment of this domain blocks this activity(Kuo et al. 2001). In vitro studies with recombinant endostatin have shown thatendostatin is able to inhibit the proliferation and migration of endothelial cells, to causeG1 arrest of endothelial cells and to induce apoptosis in certain endothelial cell lines(Dhanabal et al. 1999b, 1999c, Yamaguchi et al. 1999). Kim et al. (2000) report thatendostatin inhibits the invasion of endothelial and tumor cells into the BM by blockingthe activation and catalytic activity of MMP-2. The NC1/endostatin domain of C. eleganshomologue of type XVIII collagen has been shown to be important for cell migration andaxon guidance (Ackley et al. 2001).

2.3.4.2 Receptors for type XVIII collagen-derived endostatin

Since the soluble and immobilized forms of endostatin have different effects onendothelial cell functions in a different manner, the presence of receptors mediating thesignals of endostatin action is implicated. Cellular receptors for endostatin have beenstudied by recombinantly produced human endostatin, showing its binding to α5β1-, αvβ3- and αv β5-integrins on human endothelial cells, the binding being of functionalsignificance in vitro (Rehn et al. 2001, Sudhakar et al. 2003). Karumanci et al. (2001)have suggested in their study that glypicans are low-affinity receptors of endostatin,possibly introducing endostatin to high-affinity receptors, which affect the intracellularsignaling. Altogether, the precise molecular mechanisms of endostatin actions are stillunclear.

2.3.4.3 Molecular interactions of type XVIII collagen

In addition to the suggested binding to receptors, interactions with other molecules havealso been studied. Type XVIII collagen is localized to, and is a component of BM, and itsinteractions with other extracellular matrix molecules have been studied (Sasaki et al.1998, Miosge et al. 1999, Sasaki et al. 2000, Javaherian et al. 2002). C-terminalfragments of type XVIII collagen, NC1 and endostatin, bind fibulin-1, fibulin–2,nidogen-2 and laminin-1, and the NC1 domain also binds perlecan (Sasaki et al. 1998,

27

Miosge et al. 1999, Javaherian et al. 2002). A recent report suggested compensationbetween type XVIII collagen and perlecan heparan sulfate side chains in a study withmutant mice lacking three heparan sulfate side chains of perlecan molecule and thedouble mutant mice generated from the perlecan mutant mouse line and type XVIIIcollagen null mouse line (Rossi et al. 2003). Type XVIII collagen interacts with L-selectin, a molecule expressed on leukocytes and lymphocytes and with monocytechemoattractant protein-1 (MCP-1), and is suggested to provide a link between selectinmediated cell adhesion and chemokine-induced cellular activation, thus affectingleukocyte infiltration during inflammation (Kawashima et al. 2003). Type XVIII collagenis also suggested to have an important role in neuromuscular synapse formation in C.elegans (Ackley et al. 2003). The endostatin fragment of type XVIII collagen binds alsoto laminin-1 and to heparan sulfates on the cell surface, suggesting that the endostatin oftype XVIII collagen may provide a bridge between the BM and the cell surface (Sasaki etal. 1998, Javaherian et al. 2002). Endostatin also binds to zinc cation and heparin in vitro,and the importance of these bindings to the actions and structure of endostatin have beendiscussed (Boehm et al. 1998, Ding et al. 1998, Sasaki et al. 1999, Yamaguchi et al.1999, Hohenester et al. 2000).

2.3.4.4 Human disease of defective type XVIII collagen

Defects in human type XVIII collagen result in Knobloch syndrome characterized byoccipital encephalocele and various eye defects such as high myopia, macularabnormalities, vitreoretinal degeneration and retinal detachment (Sertié et al. 2000,Suzuki et al. 2002). All patients have ocular abnormalities usually leading to bilateralblindness, but clinical variability is present (Knobloch and Layer, 1971, Czeizel et al.1992, Seaver et al. 1993, Passos-Bueno et al. 1994, Wilson et al. 1998, Snidermann et al.2000, Sertié et al. 2000, Suzuki et al. 2002, Kliemann et al. 2003). A splice site mutationof the type XVIII collagen gene was identified leading to loss-of-function of the shortform of this collagen (Sertié et al. 2000). Other mutations in the type XVIII collagen genelead to deficiency of either the short or all isoforms of type XVIII collagen (Suzuki et al.2002). Mutations causing the abolishment of all the isoforms of type XVIII collagenpresent more severe ocular alterations compared to the lack of only the short form of thiscollagen (Suzuki et al. 2002). To date, no other human phenotype has been reported to becaused by mutations in the type XVIII collagen gene.

2.3.4.5 Mice lacking the type XVIII collagen

A mouse model has been established to clarify the function of type XVIII collagen and tostudy the consequences of the lack of type XVIII collagen in Col18a1 null mice (Fukai etal. 2002). In the light of the ubiquitous localization of type XVIII collagen, the micelacking this collagen were surprisingly healthy, viable and reproducing well. However,

28

abnormalities in the outgrowth of retinal vasculature and delayed regression of hyaloidcapillaries were observed, suggesting a vital role for type XVIII collagen/endostatin inthe development of the eye vasculature (Fukai et al. 2002). The vitreal matrix separatesfrom the inner limiting membrane of retina in the Col18a1 null mice, and as type XVIIIcollagen is localized to sites where collagen fibrils of vitreous connect to the innerlimiting membrane, it has been suggested that type XVIII collagen is important foranchoring the vitreal collagen fibrils to the inner limiting membrane (Fukai et al. 2002).The findings provide an explanation for the vitreoretinal degeneration, retinal detachmentand high myopia seen in patients with Knobloch syndrome caused by defective typeXVIII collagen (Fukai et al. 2002). Since no skull defects could be detected in theCol18a1 null mice, the localized defect in the occipital region of the skull is considered tobe human-specific (Fukai et al. 2002). At the time of the writing process of this thesis, anarticle has appeared describing dispersed pigment and abnormalities in the retina and irisin type XVIII collagen null mice (Marneros & Olsen 2003).

2.4 The eye

Perhaps the most important sensory organ for humans is the eye, delivering informationfrom the outside world by converting light into electrical impulses that are transported viathe optic nerve to the central nervous system for interpretation and for possible responseto the information. In this section, the development and structure of the human and mouseeye are reviewed.

2.4.1 The structure and development of the eye

The eyeball (bulbus oculi) fills the bony cavity, orbita, together with adipose tissue. Theeye is composed of three layers: the retina as the innermost layer, the uvea in the middleand the sclera-cornea as the outermost layer. In addition, three cavities exist in the eye,namely the anterior and posterior chambers and the vitreous cavity. A schematicpresentation of the eye structure is presented in Fig. 2. Six extrinsic eye muscles providemovement to the eyeball (Leeson et al. 1998, Sadler 2000, Kierszenbaum 2002).

2.4.1.1 Development of the eye

During the embryonic development the optic vesicles are formed from neuroectoderm tothe lateral sides of the diencephalon region of the developing brain, remaining connectedto the brain by an optic stalk. The eye is derived from embryonic surface ectoderm,neuroectoderm and mesenchyme. The surface ectoderm of the head forms the lensplacode on the optic vesicle and invaginates into it to form the lens vesicle, whicheventually separates from the ectoderm. Mesenchyme surrounds both the optic and lens

29

vesicles and also becomes the vitreous component of the eye. The optic vesicleinvaginates to form a double-layered optic cup, where the inner layer cells proliferate toform the neural retina and the outer cell layers form the pigmented retinal epitheliumcells. The outermost surface of the eye primordium is surrounded by loose mesenchyme,which differentiates into two layers, the inner layer comparable to the pia mater of thebrain and giving rise to highly vascularized choroid, whereas the outer layer developsinto sclera, which is continuous with the dura mater surrounding the optic nerve andcontinues anteriorly to form the stromal part of the cornea. (Graw 1996, Fini et al. 1997,Oliver & Gruss, 1997, Sadler 2000.)

2.4.1.2 Sclera-cornea

The sclera is the outermost layer of the eye containing collagen and elastic fibers that areproduced by fibroblasts, and the tendons of eye muscles are attached to the surface ofsclera. It is separated from the underlying choroid by loose connective tissue and anelastic tissue network, the suprachoroid lamina. The cornea is transparent, avascular andcontains a great number of free nerve endings. The cornea consists of five layers, thesurface epithelium, the membrane of Bowman, the central stromal layer containing e.g.collagen, Descemet’s membrane and the inner endothelial layer facing the anteriorchamber (Fig. 2). The cornea has a great wound healing capacity and due to its lack ofblood and lymphatic vessels, it can be successfully replaced by transplantation. The outersurface of the cornea is kept constantly wet by a film of tears that is retained by microvilliof the apical epithelial cells. (Leeson et al. 1988, Kierszenbaum 2002.)

2.4.1.3 Uvea

The vascularized part of the eye forms the uvea, and it comprises the middle layer of theeye. Uvea consists of the choroidal layer, the uveal portion of the ciliary body and theanterior part of the iris. The choroid is a vascular layer covering the posterior two thirdsof the eye. The ciliary body and its ciliary processes that extend inwards are locatedanteriorly. The uveal part of the ciliary body is continuous with the choroid. Thevascularized layer continues as the iris that contains capillaries in its anterior stromal part,whereas the posterior part of iris is epithelial. The uvea can be affected by severalinflammatory processes known as uveitis. Uveitis can target the choroid (choroiditis), theciliary body (cyclitis) and the iris (iritis). (Leeson et al. 1988, Tran et al. 2000,Kierszenbaum 2002.)

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2.4.1.4 Iris

The most important structure that regulates the amount of light entering the eye inaddition to the eyelids is the iris, which dilates (mydriasis) and contracts (miosis)according to the intensity of light in the surroundings and neural signals. The irisstructure is schematically highlighted in Fig. 2. The iris has an anterior stromal part thatcontains melanocytes, fibroblasts and capillaries. On the anterior side of the stroma thereis a discontinuous BM facing the anterior chamber. On the posterior side of the iris thereis double-layered pigmented epithelia that is separated from the stromal part of the irisby a continuous BM. A continuous BM covers also the posterior pigment epithelia on itsbasal side. The two cell layers are apically joined by gap and adherens junctions inaddition to desmosomes in the case of human and rhesus monkey (Freddo 1984). Theepithelial cells are laterally connected by tight junctions in addition to the abovementioned structures. The dilator pupillae is formed by myoepithelial extensions of theanterior pigment epithelium and it dilates the pupil in order to enable the penetration of agreater amount of light into the eye. The sphincter muscle of the iris is formed byseparate smooth muscle cells and it contracts the pupil and subsequently restricts theamount of light entering the eye. The neural signals for the dilator and sphincter muscleare provided by sympathic and parasympathic nerve fibers, respectively. (Leeson et al.1988, Freddo 1984, Freddo 1996, Kawasaki & Kardon 2001, Kierszenbaum 2002.)

2.4.1.5 Ciliary body

The innermost layer of the ciliary body is the non-pigmented epithelial cell layer that isapically joined with the pigmented epithelial cell layer (Fig. 2). A clear BM can be foundon the basal side of both epithelia. In addition to the double-layered epithelium the ciliarybody comprises vascularized stroma that contain fenestrated capillaries. The ciliary bodyis important for the maintenance of intraocular pressure (IOP), since it secretes aqueoushumour to the vitreous cavity. It is also the attachment site for the lens zonular fibers andthus affects the accommodation of the lens and plays an important role in vision. Somemolecular components of the inner limiting membrane are synthesized and secreted byciliary body epithelial cells. (Leeson et al. 1988, Jacob & Civan 1996, Bertazolli-Filho etal. 2001, Bishop et al. 2002, Escribano & Coca-Prados 2002, Kierszenbaum 2002.)

2.4.1.6 Retina

The inner layer of the eye is the retina, which is comprised of the posterior light-sensitiveportion and the anterior non-sensitive portion. The ora serrata is the border area betweenthese two zones. The light non-sensitive part of the retina consists of a single layer ofcuboidal pigmented cells that reach from the optic disc area to the ora serrata andcontinue as the pigmented epithelium of the ciliary and iris. The neural retina consists of

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photoreceptor cells and a group of neural cells: astrocytes, bipolar cells, amacrine cellsand ganglion cells residing in the inner part of the retina. Axons from ganglion cellsextend along the inner limiting membrane to the papilla region to form the optic nerve.The inner sensory retina extends from the edge of the optic disc to the ciliary epithelium.Two anatomically distinctive areas can be found in the human sensory retina: the foveacentralis is the area of the retina where vision is the sharpest, and the macula lutea is theyellow region surrounding the fovea centralis. (Leeson et al. 1988, Kierszenbaum 2002.)

Fig. 2. A schematic presentation of adult mouse eye. Detailed structures of the cornea, the iris,the ciliary body and the Bruch’s membrane are presented in the inserts. The Bowman’s andDescemet’s membranes of the cornea are indicated by white arrows. The BM layers of the irisand the ciliary body are indicated by white arrows. The lamina lucidas and lamina densas ofthe Bruch’s membrane are indicated by white arrows and arrowheads, respectively. L, lens; V,vitreous; R, retina; C, cornea; Cb, ciliary body; Ch, choroid; I, iris; S, sclera, Ep, epithelia; St,stroma; En, endothelium; AME, anterior myoepithelium; PPE, posterior pigment epithelium;A, anterior chamber; P, posterior chamber; E, nonpigmented epithelium; PE, pigmentedepithelium; RPE, retina pigment epithelium; m., membrane.

2.4.1.7 Cavities of the eye

The anterior chamber is located between the cornea and the anterior part of the iris,whereas the posterior chamber is situated between the lens and the posterior part of theiris. The vitreous cavity fills the space from the lens to the retina and contains vitreousliquid (aqueous humour). The vitreous liquid contains various proteins (such as collagens,

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see below), hyaluronic acid, ions, glucose, ascorbic acid and mainly water (98%). Theaqueous humour is produced by the ciliary body epithelium lining the ciliary processes,and some of the molecules are produced by hyalocytes. The vitreous liquid maintains theintraocular pressure in the eyeball and transports nutrients to the avascular cornea,whereafter it drains out mainly through the Schlemm’s canal (85%) and to a minor extentthrough the connective tissue surrounding the muscle fibers of the ciliary body(uveoscleral pathway) to the venous system of the eye. (Leeson et al. 1988, Jacob &Civan 1996, Kierszenbaum 2002.)

2.4.1.8 Vasa hyaloidea propria (VHP) and tunica vasculosa lentis (TVL)

During the development and maturation of the human and mouse eye, the hyaloid vesselsundergo regression. The vessels disappear in humans before birth, and in mouse theyregress in a short period of time after birth (Fig. 3). The VHP is attached to the initiallyavascular retina providing it with oxygen and nutrients. After birth the mouse VHPdetaches from the retina and the hyaloid capillaries are pushed towards the lens by theformation of secondary vitreous. In the VHP and TVL, the regression proceedssegmentally and results in a decreased number of capillary interconnections. Eventuallythe mouse VHP disappears by the age of 12 to 16 days and no vitreal capillaries can bedetected in normal adult mice, while some examples of TVL remain even at 16 days. Theregression of VHP and TVL is suggested to take place via apoptosis, and hyalocytes/macrophages (Lazarus & Hageman 1994), cells belonging to leucocyte-lineage (Zhu etal. 1999), are likely to play a central role in the hyaloid vessel regression pathway (Lang& Bishop 1993). (Jack 1972, Ko et al. 1985, Ito & Yoshioka 1999, Zhu et al. 2000.)

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Fig. 3. A schematic presentation of the regression of mouse VHP and TVL at four postnataltimepoints (arrows and stars, respectively). L, lens; R, retina.

2.4.2 Does the mouse eye differ from the human eye?

Animal models have been generated in order to understand better the molecularmechanisms behind diseases. The mouse is an ideal species for this purpose for manyreasons, one of the most important being the homology between the genome of mouseand man. The mouse reaches its adulthood in quite a short time span, so the study of adultstages is possible within a reasonable time period. The living environment of mice canalso be well controlled and the access of pathogens restricted. Despite the similaritiesbetween men and mice, a mouse is still a mouse and differences exist compared toprimates. In the eye the most striking difference is the size. Both species have the samestructures in the eye, except for the macula, which is lacking in the mouse. The lens isstrikingly large in the mouse, filling up most of the vitreous cavity, and subsequently theamount of vitreous is relatively smaller compared to human. In primates there is a large

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triangular bed of smooth muscle in the ciliary region. This ciliary muscle is very small inmice and may have little function. Only a few muscle cells are found external to ciliaryprocesses. Although the overall structure of the trabecular meshwork is identical betweenman and mouse, the number of the trabecular beams is smaller and the termination in thearea of anterior ciliary body is slightly different in mouse compared to human. Many ofthe mouse strains are albinos, but the only difference between them and pigmented miceis the absence of melanosomes, otherwise they are anatomically similar. (Smith et al.2000a.)

2.4.3 Collagens of the eye

Several members of the large protein family of collagens can be found in the eye. Thehemidesmosomes connecting epithelial cells to the underlying BM typically contain typeVII and XVII collagens in the cornea and limbus (Gipson et al. 1987, Gipson 1989, Tuoriet al. 1996, Gordon et al. 1997). Collagens II, IX and V/XI can be found in the vitreousof several species (Linsenmayer et al. 1982, Fitch et al. 1988, Marshall et al. 1993,Mayne et al. 1993, Bishop et al. 1994, Azuma et al. 1998, Bishop et al. 2002) and alsotype III collagen can be found in the developing human vitreous, where it is entirelyreplaced by collagen II during embryonic development (Azuma et al. 1998). Type IVcollagen is typically found in BMs of the eye (Rodrigues et al. 1980, Benezra & Foidart1981, Konstas et al. 1990, Marshall et al. 1993, Fukuda et al. 1999, Halfter et al. 2000,Kelley et al. 2002, Chen et al. 2003). Also collagens I, III, VI, VIII, XII, XIV, XVIII,XX, XXIV, XXVII are localized to the eye (Kapoor et al. 1988, Zimmermann et al. 1988,Konstas et al. 1990, Rittig et al. 1990, Marshall et al. 1993, Gordon et al. 1996, Koch etal. 2001, Lin HC et al. 2001, Meek & Fullwood 2001, Akimoto et al. 2002, Elamaa et al.2002, Young et al. 2002, Kato et al. 2003, Koch et al. 2003, Pace et al. 2003).

2.4.4 Laminins of the eye

Laminins are extracellular matrix molecules consisting of the α, β and γ chain. Theseglycoproteins provide structural support in animal tissues, and through interaction withvarious molecules they also affect cell migration and differentiation as well as cell andtissue survival. Laminins can bind to other matrix molecules and interact with cellsthrough integrins, dystroglycan and other receptors. 15 different combinations ofheterotrimeric laminins have been characterized so far (Colognato and Yurchenco, 2000,Libby et al. 2000). There are several studies on laminin chain expression in thedeveloping and adult eye (Ljubimov et al. 1995, Libby et al. 1996, Libby et al. 1997, Qinet al. 1997, Falk et al. 1999, Libby et al. 2000, Zhang et al. 2001). The expression ofdifferent laminin polypeptides in rat and human retina has previously been studied byLibby et al. (1996, 2000). Their study includes the expression of the three chains formingthe laminin-5 molecule, namely the α3, β3 and γ2. They showed that the laminin α3chain is present in the interphotoreceptor matrix, external limiting membrane and outerplexiform layer, whereas the β3 chain expression in the retina is limited only to the

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interphotoreceptor matrix. Also the laminin γ2 chain was shown to be present ininterphotoreceptor matrix in rat and human, and in hyaloid vessels and, to a limitedextent, the intraretinal capillaries in human (Libby et al. 2000). Qin et al. (1997) havestudied the expression of laminin-1, laminin-5 and laminin α2 chain in the developingmouse eye, and Ljubimov et al. (1995) have studied the expression of different lamininchains in the cornea. Laminin-5 is a part of this thesis study and will thus be described inmore detail.

2.4.4.1 Laminin-5

Laminin-5 (also called kalinin/nicein/ladsin/epiligrin) is a heterotrimer consisting of α3,β3 and γ2 chains (Ryan et al. 1994, Gerecke et al. 1994, Vailly et al. 1994, Utani et al.1995, Kallunki et al. 1992, Airenne et al. 2000, Colognato and Yurchenko 2000). Aschematic structure of the laminin-5 molecule is presented in Fig. 4. The α3 chain oflaminin-5 can exist either as a short 5A form, or as a long 5B form (Garbe et al. 2002). Itis an epithelium-specific laminin subtype and a component of epithelial anchoring systemof the BM interacting with hemidesmosomal integrin α6β4 and type VII collagen(Niessen et al. 1994, Rousselle et al. 1997, Aumailley et al. 2003). Proteolytic processingof laminin-5 was first identified by Rousselle et al. (1991) and subsequently characterizedby Marinkovich et al. (1992). Further studies have revealed that the γ2 chain of laminin-5is processed by bone morphogenetic protein-1 (BMP-1, Amano et al. 2000), matrixmetalloproteinase-2 (MMP-2, gelatinase A, Giannelli et al. 1997), membrane type 1-MMP (MT1-MMP, MMP-14, Koshikawa et al. 2000, Pirilä et al. 2003) and other MMPs,such as MMP-3, -13, and –20 (Pirilä et al. 2003). The α3 chain of laminin-5 is alsoproteolytically processed undergoing two cleavages and BMP-1 may have a role in thisprocess (Amano et al. 2000). The β3 chain of laminin-5 is also processed by MT1-MMP(Udayakumar et al. 2003). Laminin-5 has been found to be covalently linked to laminins6 and 7 (Champliaud et al. 1996), but the role of the complexes is unclear, although it hasbeen proposed that the complex is competent to participate in BM assembly. Defects inany of the three chains of laminin-5 result in the lethal blistering condition Herlitz’sepidermolysis bullosa (Aberdam et al. 1994, Pulkkinen et al. 1994a, b, Kivirikko et al.1995b, Ryan et al. 1999).

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Fig. 4. Chain structures of laminin 5A and 5B molecules (according to Aumailley et al. 2003).Chain domains are indicated (I-VI, G). The processing sites for MMP-2 and BMP-1 in the γ2chain are indicated (black and white arrow, respectively).

2.4.5 Anterior segment disorders

Several disorders affect one or several structures in the anterior part of the eye. Theseinclude the Axenfeld-Rieger syndrome, Peters’ anomaly, the iridogoniodysgenesisanomaly, the iridogoniodysgenesis syndrome, iris hypoplasia, the pigment dispersionsyndrome, anterior segment dysgenesis and familial glaucoma iridogoniodysplasia.(Shields et al. 1985, Alward 2000, Amendt et al. 2000, Shields 2001, Gould & John2002.) Many of these diseases have overlapping clinical features, such as iris hypoplasia,displaced Schwalbe’s line and defective trabecular meshwork, and they often associatewith glaucoma. Many gene defects behind these disorders have been identified,occupying several genes on several chromosomes, and overlapping phenotypes resultfrom mutations in different genes. Characterization of the underlying mutations hasshown that different mutations in one gene can cause different degrees of abnormality,and great variability can be observed even in the phenotypes occurring within a singlefamily. The mutations causing the Axenfeld-Rieger syndrome, for example, have beenmapped to 4q25, 6p25, 13q14 and quite recently to chromosome 11 and patients withPeters’ anomaly have abnormalities such as deletions in chromosomes 4, 11 and 18,translocation between chromosomes 2 and 15 and a ring chromosome 21. (Alward 2000,Gould et al. 1997, Riise et al. 2001, Lehman et al. 2002, Lines et al. 2002, Traboulsi

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1998.) Mutations in the transcription factor-encoding genes FKHL7/FOXC1, at 6p25(Mears et al. 1998, Nishimura et al. 1998, Mirzayans et al. 2000, Smith et al. 2000b,Kawase et al. 2001, Suzuki et al. 2001, Borges et al. 2002, Honkanen et al. 2003),PITX2/RIEG1, at 4q25 (Kulak et al. 1998, Doward et al. 1999, Priston et al. 2001,Borges et al. 2002, Phillips 2002), and PAX6, at 11p13 (Jordan et al. 1992, Azuma et al.1999, Sonoda et al. 2000), have been demonstrated in several of the disorders, but otheraffected genes remain to be identified in various associated loci (Alward 2000, Traboulsi1998). Pupillary disorders are reviewed by Kawasaki & Kardon (2001). Recentlyidentified ocular disease mutations are summarized by Haider et al. (2002).

2.4.5.1 Mouse models for anterior segment defects

Mice representing phenotypes of human anterior eye segment disorders have beengenerated and the transgenic models for eye malformations have been reviewed by Götz(1995). More recently, other mouse models for human anterior eye defects have beenpresented, including suggested models for Peters’ anomaly (Ormestad et al. 2002),pigment dispersion syndrome (John et al. 1998), congenital cataract (Graw 1999) andanterior segment dysgenesis (Chang et al. 2001). The mouse mutation Small eye issuggested to be a model for human aniridia (Hill et al. 1991, Glaser et al. 1992).

A useful web site for overviewing mouse models also for anterior eye segment defectsis the Informatics homepage of The Jackson Laboratory, (http://www.informatics.jax.org/)that provides updated information on mouse genetics.

2.5 Basement membranes – active non-cellular entities of tissues

Basement membranes form dense, sheet-like structures at the interface between epithelialand mesenchymal cells, and they are spatially and temporally unique. Some cell types,such as muscle and Schwann cells, are surrounded by BM. BMs provide structuralsupport to the overlying cells, function as active barriers for infiltration and cellmigration, and they also affect various biological activities in tissues. BMs are involved inembryonic development, wound healing, tissue remodeling and tumour metastasis.(Engvall 1995, Timpl 1996, Schwarzbauer 1999, Miosge 2001.) The BM is divided intothree layers as viewed from the epithelia: the lamina lucida, the electron-dense laminadensa and lamina fibroreticularis (for review, see Miosge 2001). The lamina lucida layerhas been proposed to be an artefact due to poor fixation (Chan & Inoue 1994). Althoughthe BM components are present in the morulae stage of embryonic development, novisible BM exists at this point. The first BM, Reichert’s membrane, is found at theblastocyst stage (for review, see Miosge 2001). The components of BMs can be groupedinto intrinsic and extrinsic ones, the former group comprising members of the lamininand collagen superfamilies and heparan sulfate proteoglycans, while the latter refers tomolecules that may be selectively incorporated into BMs and have biological importance

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to BMs, such as growth and differentiation factors (Engvall 1995). The moleculescomprising the structure of BMs appear as monomers or polymers. A remarkable varietyof BM components including laminins, perlecan, agrin, nidogen, fibulin-1 and -2 andcollagen types IV, XV, XVIII and XIX have been characterized (for reviews, see Kühn1994, Timpl, 1996, Myers et al. 1997, Schwarzbauer 1999, Amenta et al. 2000, Ericksonand Couchman 2000, Miosge 2001, Quondamatteo 2002). BMs undergo remodeling anddegradation as a response to various biological events in their surroundings, and a balancebetween the synthesis and degradation of ECM is important in BM formation (Amano etal. 2001). Matrix metalloproteinases (MMPs) are a group of peptidases that have animportant role in this modeling of BMs (Birkedal-Hansen 1995).

3 Outlines of the present study

In the beginning of this thesis study, the first cDNA clones encoding the α1 chain ofmouse type XVIII collagen had been found. However, neither the primary structure northe tissue distribution of the corresponding human polypeptide was known. As the proteinhad been newly identified, there was no information about the function or molecularinteractions of type XVIII collagen. When parts II–IV of this thesis work were started, amouse line lacking type XVIII collagen had been produced by homologousrecombination in Prof. Björn Olsen’s group at the Department of Cell Biology in HarvardMedical School, Boston, USA. In collaboration with Prof. Olsen these mice were madeavailable for us for further analysis. Based on the first eye findings in the type XVIIIcollagen null mice, the eyes of the null mice were further examined resulting in reports IIand IV. A mouse model lacking homologous type XV collagen had also been generated(Eklund et al. 2001). Both the type XVIII and XV single null mouse phenotypes weresurprisingly mild, suggesting that the two collagens may compensate for each other in thesingle null mice. Thus, the double null mice lacking both collagens was generated andstudied leading to report III in this thesis.

The aims of my thesis research were:1. To characterize the primary structure of human type XVIII collagen and elucidate of

the tissue distribution of the corresponding mRNA. 2. To further study the consequences of the lack of type XVIII collagen in the mouse eye

concentrating on the anterior eye segment.3. To study the basement membranes in the type XVIII collagen null mouse eyes.4. To study the expression of type XV and XVIII collagens in the postnatal mouse eye.5. To study phenotypic consequences of the lack of both collagens XV and XVIII in

mouse in comparison to the type XV and XVIII single null mice.

4 Material and methods

Detailed descriptions of the material and methods used in the studies can be found in theoriginal articles I–IV.

4.1 Characterization of the human α1(XVIII) cDNA clones (I)

4.1.1 Isolation of the cDNA clones

The first PCR fragment of human α1(XVIII) cDNA was obtained from human placentacDNA using primers based on the sequences of mouse type XVIII collagen (Rehn et al.1994). Bluescript SK (Stratagene) was used as a vector and the 455-bp PCR fragmentwas cloned into the EcoRI site of the plasmid. The first cDNA clone was 32P-labeled andused as a probe for screening a human placenta cDNA library (HL1075b, Clontech) andthe subsequent cDNAs were subcloned as above. MG63 osteosarcoma cell line was usedto construct a cDNA library for the isolation of additional clones. A primer-extendedcDNA pool was prepared from 2 µg of the MG63 cell poly(A)RNA using the humanα1(XVIII) collagen-specific primer JR2-RT and the Time-Saver cDNA synthesis kit(Pharmacia LKB Biotechnology Inc.). λgt10 vector (Stratagene) was used and the cDNAwas packed into bacteriophage λ particles using the in vitro packaging extract (AmershamCorp.) This library was screened with a 32P-labeled fragment of mouse type XVIIIcollagen under low stringent conditions (Sambrook et al. 1989). The subsequent positivecDNA clone L16A was subcloned into a NotI site of Bluescript SK and was used toscreen a human fetal liver cDNA library (HL1064a, Clontech) resulting in isolation ofseveral positive recombinant phages. A synthetic oligonucleotide was derived from one ofthese clones, FL7.1.1, and used to screen a human adult kidney cDNA library (HL 1123b,Palo Alto, CA, USA), from which a positive clone, HK16.2, was recovered. A 100-bpinsert of this clone was used to screen a human adult liver cDNA library (HL 1115b,Clontech, Palo Alto, CA, USA) resulting in isolation of clones HL4 and HL8. A 166-bpinsert from the HL4 was used to rescreen the human liver cDNA library resulting in

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isolation of the clone HuL8.2. A 264-bp insert of this clone was used to screen the samelibrary resulting in the cDNA clone HLi12.1. A PCR fragment corresponding to the 5’sequences of the common region of human type XVIII collagen was generated from theHL4 cDNA clone and used to screen a human fetal kidney cDNA library (HL 5004b,Clontech, Palo Alto, CA, USA) resulting in isolation of the clone HFK1.1.

4.1.2 Sequence analysis of the cDNA clones

The isolated cDNA clones were sequenced either manually or with an automated ABIDNA sequencer (Applied Biosystems). Sequenase enzyme (United States Biochemical)was used in the reactions based on the dideoxynucleotide chain termination method(Sanger et al. 1977). The subsequent nucleotide and corresponding amino acid sequenceswere analyzed with DNASIS and PROSIS (Pharmacia Biotech Inc.) and comparisons atthe nucleotide and amino acid sequence level were made against the Genbank, EMBLPIR and SWISSPROT DATABASES at the National Center for BiotechnologyInformation (NIH, USA) using the BLAST network service (Altschul et al. 1990).PROSITE database (Bairoch 1992) was used to search for structural motifs and theprediction of signal sequence was done with the SignalPV1.1 World Wide WebPrediction server (Nielsen et al. 1997).

4.1.3 Reverse transcription (RT) and polymerase chain reaction(PCR) of human cDNA

A primer-extended cDNA pool was prepared from human fetal liver total RNA (ClontechLaboratories Inc.) in order to study further the alternative splicing detected in cDNAclone HL4. 5 µg of total RNA was synthesized into single-stranded DNA in a 20 µlreaction First Strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 10mM DTT, 0.2 mM of each deoxynucleotides, 50 pmol of random hexamer primers, 20 Uof Rnasin (Promega) and 200 U murine MVL reverse transcriptase (Gibco BRL) andincubated at 37°C for 1 hour.

PCR reactions with human α1(XVIII) specific primers flanking the spliced area wereperformed using 5 µl of the above mentioned RT reactions or 5 µl of cDNA pools prepa-red from human HT-1080 cell line, jejunum or skin poly(A) RNAs with oligo(dT) primers(a generous gift from Dr. Pasi Hägg, University of Oulu, Oulu, Finland). A PCR reactionwithout a template was used as a negative control and all of the PCR products were sepa-rated on a 6% polyacrylamide gel (Sequagel-6, National Diagnostics).

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4.2 Northern blot analysis of human α1(XVIII) transcripts (I)

Tissue distribution of the human type XVIII collagen mRNAs were studied by Northernblot analysis. 32P-labeled cDNA fragments from clones HL8, HuL8.2 and HFK1.1 wereused to hybridize human adult and fetal Multi-Tissue Northern blots (Clontech, Palo Alto,CA, USA). A probe for β-actin was supplied by the manufacturer and used as a positivecontrol. The hybridization was performed according to the manufacturer’s protocol. AcDNA fragment corresponding to the spliced region of human cDNA was used as a probein some experiments.

4.3 Type XVIII collagen null mice (II, IV)

The mouse line lacking type XVIII collagen was generated by homologousrecombination in Prof. Björn Olsen’s group (Department of Cell Biology, HarvardMedical School, Boston, USA) as described (Fukai et al. 2002).

4.4 Type XV and XVIII double null mice (III)

The mouse line lacking both type XV and XVIII collagen was generated by breeding typeXV single null mice (Eklund et al. 2001) and type XVIII single null mice (Fukai et al.2002). The genotype of the mutant mice was confirmed by Southern blotting and PCR.The downstream PCR primer for the intact and Col18a1 null allele was 5’-CTCTGTAGGGTCCTTATGGACG-3’, and the upstream primers were 5’-CATTCGTTCCAGGTCGACCCTG-3’ and 5’- AGGAGTAGAAGGTGGCGCGAAGG-3’ for the wild-type and null alleles, respectively. The upstream PCR primer for the wild-type and Col15a1 null allele was 5’-AGGGGAACGAGAGTTCAA ATCCATAGA-3’,and the downstream primers were 5’-TGGCAGTGTGTTGTCACT GTACAGCTA-3’ and5’-CGCCTCATTCCTGGACATAAAAGGG-3’, respectively.

4.5 Histological studies of mouse eyes (II–IV)

4.5.1 Sample preparation

Paraffin sections were prepared by immersion fixing of the eyes in phosphate-bufferedformalin overnight at room temperature. The penetration of the fixative was ascertainedby making one or more holes on the back of the globe with a needle or knife. Thesamples were embedded in paraffin wax using routine methods, and 5 µm, or in the caseof adult eyes 10 µm, sagittal sect