Contribution of the lysyl hydroxylase 2 and lysyl oxidase enzymes to the remodeling of the extracellular matrix. Implications in the biosynthesis of fibrillar collagen and its application in tissue engineering

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UNIVERSIDAD AUTÓNOMA DE MADRID

PROGRAMA DE DOCTORADO EN BIOCIENCIAS MOLECULARES

Contribution of the lysyl hydroxylase 2 and lysyl oxidase enzymes to the

remodeling of the extracellular matrix. Implications in the

biosynthesis of fibrillar collagen and its application in tissue engineering.

Tamara Rosell García

Madrid, 2020

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Universidad Autónoma de Madrid

Facultad de Ciencias

Departamento de Biología Molecular

CONTRIBUTION OF THE LYSYL HYDROXYLASE 2 AND LYSYL OXIDASE ENZYMES TO THE REMODELING OF THE EXTRACELLULAR MATRIX.

IMPLICATIONS IN THE BIOSYNTHESIS OF FIBRILLAR COLLAGEN AND ITS APPLICATION IN TISSUE ENGINEERING.

MEMORIA

para optar al grado de Doctora en Biociencias Moleculares presentada por:

TAMARA ROSELL GARCÍA

Licenciada en Biología

DIRECTOR:

Fernando Rodríguez Pascual

Científico Titular

TUTORA:

Susana Cadenas Álvarez

Científico Titular

Centro de Biología Molecular “Severo Ochoa”, CSIC

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Fernando Rodríguez Pascual, Doctor en Química, Científico Titular del CSIC en el

Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas)

CERTIFICA:

Que Doña Tamara Rosell García, Licenciada en Biología por la Universidad Autónoma de Madrid, ha realizado bajo su dirección la presente Tesis Doctoral titulada

“Contribution of the lysyl hydroxylase 2 and lysyl oxidase enzymes to the remodeling of the extracellular matrix. Implications in the biosynthesis of fibrillar collagen and its application in tissue engineering”, y que dicho trabajo reúne los requisitos necesarios

para su presentación y defensa pública para optar al grado de Doctor por la Universidad Autónoma de Madrid.

Y para que conste firmo como director,

Fdo: Dr. Fernando Rodríguez Pascual

Madrid, junio de 2020

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A mis padres A mi abuela, porque nunca necesité escucharte decir “Te quiero”

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“Defiende tu derecho a pensar, porque incluso pensar de manera errónea

es mejor que no pensar”

Hipatia de Alejandría

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Agradecimientos

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Agradecimientos

III Agradecimientos

La presente Tesis Doctoral, realizada en el Centro de Biología Molecular “Severo Ochoa”

(CBMSO) del Consejo Superior de Investigaciones Científicas (CSIC), ha sido cofinanciada por los proyectos “Role of the lysyl oxidase family in the pathogenesis of connective tissue diseases:

contribution of the aortic alterations of Marfan syndrome and related disorders” (SAF2012- 34916) y “Role of the lysyl oxidase family in the development of cardiovascular diseases:

Analysis of mechanistic insights and therapeutic options” (SAF2015-65679-R) del programa Proyectos de Investigación Fundamental no orientada así como por el proyecto I+D+i “Retos Investigación” del Programa Estatal de I+D+i orientada a los retos de la sociedad “Activación proteolítica de lisil oxidasas. Aplicaciones biotecnológicas y contribución al remodelado de la matriz extracelular en el desarrollo de enfermedades humanas” (RTI2018-095631-B-I00).

Todo trabajo de investigación requiere de la participación de numerosos agentes, personas e instituciones para que llegue a feliz término. Esta tesis doctoral no es una excepción.

Por ello, antes de comenzar me gustaría dedicar unas líneas para expresar mi más sincero agradecimiento a las numerosas personas que, de una manera u otra, han contribuido a la realización de esta tesis.

En primer lugar, a mi director de Tesis, el Dr. Fernando Rodríguez Pascual, por darme la oportunidad de formar parte de tu grupo de investigación sin apenas conocerme, por tu dedicación y apoyo durante estos cinco años. Gracias por permitirme trabajar mano a mano contigo y aprender de ti cada día, sabes que esta tesis es más tuya que mía. Gracias por tu rigurosidad, buen hacer y enorme capacidad de trabajo. Por siempre sobreponerte a las adversidades que nos hemos encontrado en el camino. Pero sobre todo gracias por tu amistad, por siempre estar ahí, saber escuchar y entender todas las situaciones. Trabajar contigo ha sido siempre un placer. Gracias por hacer de estos cinco años los más felices de mi carrera profesional.

A mi tutora de Tesis en el Departamento de Biología Molecular de la Universidad Autónoma de Madrid (UAM), la Dra. Susana Cadenas Álvarez, por su interés y excelente disposición.

A las Dras. Anabel Marina y Esperanza Morato, del Servicio de Proteómica del Centro de Biología Molecular “Severo Ochoa” (CBMSO) del CSIC y al Dr. Alberto Paradela y Gema Bravo, del Servicio de Proteómica del Centro Nacional de Biotecnología (CNB) del CSIC, por toda su ayuda y colaboración en los estudios de proteómica sobre la isoforma de LOX.

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Agradecimientos

IV

Al Dr. Alain Colige, de la Universidad de Liegè (Bélgica) por permitirme llevar a acabo una estancia predoctoral en su grupo de investigación. Gracias también por su colaboración en el estudio sobre la proteolisis de LOX mediada por ADAMTS2/14, así como a la Dra. Laura Dupont y el Dr. Mourad Bekhouche.

Al Dr. Santiago Lamas, por compartir su laboratorio conmigo y hacerme sentir querida y acogida como una más. Gracias por permitirme compartir grandes momentos con todo tu equipo tanto dentro como fuera del laboratorio. Me habéis hecho muy feliz estos cinco años. Y por supuesto muchas gracias por tu visita durante mi estancia en Liegè, que siempre recordaré con cariño.

A mi pequeña gran familia del SLamas. Gracias por todo vuestro cariño y apoyo y por ser los mejores compañeros de laboratorio que pudiera desear. A mis unicornios favoritos Vero y Patri, gracias por ser las mejores amigas, por apoyarme siempre y hacerme sentir como en familia desde el primer momento que nos conocimos. Me siento muy afortunada de haberos encontrado y haber compartido esta experiencia con vosotras. Muchas gracias también a Macarena, por tu amistad, por todo tu apoyo y cariño siempre. Gracias chicas por un inolvidable viaje a Noruega, siempre será uno de mis mejores recuerdos. Por muchos viajes más las cuatro juntas, con o sin pandemia de por medio. A Carlitos, gracias por aceptar finalmente que eres una más y por tantas risas y buenos momentos. Gracias por tu amistad y todo el ánimo: eres la siguiente, mucha suerte.

A Jessy, la mejor lab manager del mundo mundial. Gracias por tu amistad, las tartas de tu madre y por ser un paibon como yo, ya no me siento tan incomprendida en este laboratorio lleno de adefesios. A Diana, por tu apoyo, amistad y sabios consejos en este mundo de la ciencia. Muchas gracias por las risas, viajes y buenos momentos compartidos.

A todos los compañeros que han pasado por el laboratorio y que de una manera u otra me han ayudado durante estos años: José, Mari Ángeles, mi pollito Óscar, Marta, Eva, Estrella, Nacho y todos los innumerables pollitos del SLamas.

A todos los trabajadores del CBMSO por los servicios prestados durante este tiempo.

Y lo más importante: gracias a mis padres por su amor incondicional y su dedicación continua. Mi más sincero agradecimiento por vuestro apoyo incondicional en todas las acciones que he emprendido a lo largo de mi vida. Todo lo que soy y todo lo que he conseguido ha sido gracias a vosotros.

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Summary

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Summary

IX Summary

Type I collagen belongs to a large family of extracellular matrix (ECM) proteins that play an essential role in providing form, stability and connectivity in many tissues. One of the prevalent features of collagen is its extensive post-translational modifications (PTMs), including lysine modifications, that are critical to the structure and biological functions of this protein.

Inside the cell, specific lysine residues in the telopeptides regions of the collagen molecule are hydroxylated by lysyl hydroxylase-2 (LH2), being this PTM essential for the formation of stabilized cross-links. LH2 expression has been described to be strongly regulated by transforming growth factor 1 (TGF-1) and hypoxia, well-known stimuli for the synthesis and deposition of a fibrotic ECM. Here, we have identified specific binding sites for hypoxia- inducible factors (HIF) and TGF--activated SMAD proteins in the promoter of the LH2 gene (PLOD2). We have also revealed that HIF signaling plays a preponderant role in the induction of PLOD2 expression. Moreover, we demonstrate that expression of PLOD2 is fundamental for these profibrotic stimuli to promote deposition of collagen onto the insoluble matrix.

Outside the cell, specific lysine and hydroxylysine residues in the telopeptides can be oxidatively deaminated by lysyl oxidase (LOX) to form intra- and intermolecular cross-links.

This step is also critical for the stabilization of the assembled fibrils. Several experimental evidences demonstrate the existence of different mature forms that contribute to LOX-mediated actions. Our work describes that LOX is proteolytically process by the procollagen N-proteinases ADAMTS2 and 14 (a disintegrin and metalloproteinase with trombospondin motifs), in an amino acid location downstream of the BMP1(bone morphogenetic protein 1) cleavage site. The sequence between these two processing sites contains a cluster of tyrosines that can be potentially modified by O-sulfation and contribute to the binding of the LOX enzyme to collagen.

Finally, we have focused our work in the development of cultured cell-derived ECM- based biomaterials. Standard cell culture conditions do not favor the production of ECM components. In particular, in vitro collagen deposition is severely limited due to an incomplete conversion of procollagen by BMP1. To this end, we have developed a protocol to enhance the capacity of in vitro cell cultures to deposit collagen onto the ECM based on the exogenous addition of LOX and BMP1-enriched supernatants to fibroblast cultures. We also show that these fibroblast-derived matrices are able to regulate adipogenic and osteogenic differentiation, and this effect is modulated by LOX/BMP1. Thus, this approach represents a promising technology for application in tissue engineering.

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Abbreviations

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Abbreviations

XIII Abbreviations

ADAMs A disintegrin and metalloproteinases

ADAMTS A disintegrin and metalloproteinase with thrombospondin motifs ACP Aldol condensation product

BAPN -aminopropionitrile BM Basement membrane

BMP1 Bone morphogenetic protein1 CCI4 Carbon tetrachloride

CRL Cytokine receptor-like

deH-DHLNL Dehydro-dihydroxylysinonorleucine deH-HLNL Dehydro-hydroxylysinonorleucine deH-HHMD Dehydrohistidinohydroxymerodesmosine deH-LNL Dehydro-lysinonorleucine

d-PRL Deoxypyrrole DxS Dextran sulfate ECM Extracellular matrix EDS Ehlers-Danlos syndrome

EMT Epithelial-mesenchymal transition GAGs Glycosaminoglycans

HAS HIF ancillary sequence HBS HIF binding site

HIF-1 Hypoxia-inducible factor-1 HP Hydroxylysylpyridinoline HRE Hypoxia responsive element Hyl Hydroxylysine

Hylald Hydroxyallysine =Hydroxy-α-aminoadipic acid-δ-semialdehyde Hyp Hydroxyproline

LH Lysyl hydroxylases LOX Lysyl oxidase LOXL Lysyl oxidase like LP Lysylpyridinoline LTQ Lysyl tyrosyl quinone

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Abbreviations

XIV Lys Lysine

Lys^ald Allysine = α-aminoadipic acid-δ-semialdehyde MMPs Matrix metalloproteinases

MSCs Mesenchymal stem cells mTLD Mammalian tolloid P3H Prolyl 3-hydroxylases P4H Prolyl 4-hydroxylases

PDGF Platelet-derived growth factor

PLOD Procollagen-lysine, 2-oxoglutarate 5-dioxigenase Pro Proline

PRL Pyrrole

PTMs Post-translational modifications SRCR Scavenger receptor cysteine-rich TGF-1 Transforming growth factor 1 TLL-1 Tolloid like 1

TM Tetrathiomolybdate

XSF Pseudoexfoliation syndrome

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DIFFERENTIAL CLEAVAGE OF LYSYL OXIDASE BY THE METALLOPROTEINASES BMP1 AND ADAMTS2/14 REGULATES COLLAGEN BINDING THROUGH A TYROSINE SULFATE DOMAIN. 55 ENHANCEMENT OF COLLAGEN DEPOSITION AND CROSS-LINKS BY COUPLING LYSYL OXIDASE WITH BONE MORPHOGENETIC PROTEIN-1 AND ITS APPLICATION IN TISSUE ENGINEERING. .... 85

DISCUSSION ... 105

4.1THE PROFIBROTIC STIMULI HYPOXIA AND TRANSFORMING GROWTH FACTOR 1 INDUCE PROCOLLAGEN LYSYL HYDROXYLASE 2 EXPRESSION BY MEANS OF A HIERARCHICAL NETWORK BETWEEN HYPOXIA-INDUCIBLE FACTOR AND SMAD PROTEINS ... 107

4.1.1 Hypoxia and TGF-1 responsive elements in the human PLOD2 promoter drive the expression of the PLOD2 gene. ... 108

4.1.2 PLOD2 expression driven by TGF-1 and hypoxia is key to promote collagen deposition into the insoluble ECM in vitro ... 109

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Table of contents

XVIII

4.2LOX CLEAVAGE REGULATES COLLAGEN BINDING THROUGH A TYROSINE SULFATE DOMAIN

... 112

4.2.1 LOX is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 in vitro ... 114

4.2.2 A tyrosine-sulfate domain in the LOX enzyme drives its union to the telopeptides and regulates its activity on the collagen molecule in vitro ... 115

4.3BOOSTING COLLAGEN DEPOSITION AND CROSS-LINKING IN VITRO BY COMBINING LOX AND BMP1 ENZYMES AND ITS APPLICATION IN TISSUE ENGINEERING ... 117

CONCLUSIONS ... 123

REFERENCES ... 129

APPENDIX ... 153

APPENDIX 1.1RESUMEN ... 157

APPENDIX 1.2CONCLUSIONES ... 158

APPENDIX 2 ... 161

PUBLICATIONS ... 161

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Table of contents

XIX Table of Figures

Figure 1. Composition of the ECM ... 4

Figure 2. Structure of human type I collagen ... 5

Figure3. Type I collagen biosynthesis and cross-linking ………...6

Figure 4. Mayor cross-linking pathways of collagen ..……..………...…10

Figure 5. Domain organization of members of the lysyl oxidase (LOX) famiy…………...…….12

Figure 6. Modulation of the in vitro microenvironment using macromolecular crowding to imitate the in vivo dense extracellular space……….….21

Figure 7. Hypoxia- and TGB-dependent PLOD2 expression…………...………111

Figure 8. Proposed model describing the regulatorymechanisms controlling LOX biological activity in the context of collagen processing and cross-linking………..113

Figure 9. Schematic representation of the protocol developed to poteniate collagen deposition in fibroblasts cultures………..………118

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Introduction

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Introduction

3 1. Introduction

1.1 The extracellular matrix

The extracellular matrix (ECM) constitutes an intricate molecular network that surrounds and integrates cells and tissues in multicellular organisms. In addition to providing tissues with their biomechanical properties, it also influences important cellular processes (1,2) including cell proliferation, adhesion, migration and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. Far from being a static material, the ECM is a highly dynamic structure, constantly undergoing a remodeling process whereby constituents are deposited, degraded, or modified. These molecular components are subjected to a myriad of post- translational modifications (PTMs). Tightly controlled ECM homeostasis is essential for development, wound healing and normal organ homeostasis. Hence, sustained dysregulation can result in life-threatening pathological conditions. The importance of correct biochemical and biophysical ECM properties such as composition, structure, stiffness or abundance on the regulation of cell and tissue homeostasis is illustrated by the fact that the ECM is unregulated in many types of conditions such as cancer and cardiovascular or fibrotic diseases. A better understanding of the regulation of ECM remodeling and how affects disease progression is, therefore, essential for the development of new therapeutic strategies in tissue engineering and regenerative medicine.

1.1.1 ECM components

The ECM is mainly composed of an intricate interlocking mesh of fibrillar and non- fibrillar collagens, elastic fibers and glycosaminoglycan (GAG)-containing non-collagenous glycoproteins (hyaluronan and proteoglycans) (Figure 1). All these diverse molecules are synthesized and secreted locally by specialized cells predominantly belonging to the mesenchymal lineage (e.g. (myo)fibroblasts, chondrocytes, osteoblasts, tenocytes). There are two main types of ECM that differ with regard to their location and composition: the interstitial matrix and the basement membrane. The interstitial matrix surrounds and supports cells, and provides structural scaffolding to the tissues. It also acts as a compression buffer for tissues subjected to deforming stress. This matrix is found in most tissues and consists mainly of collagen I, which, together with fibronectin, confers mechanical strength to tissues. In addition, the basement membrane (BM) is a specialized form of ECM that separates the epithelium from the surrounding stroma. It mainly consists of collagen IV, laminins, entactin and heparan sulfate proteoglycans.

BMs play a key role in epithelial cell function, providing cues for orientation that help establish and maintain apicobasal polarity and cell differentiation

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. However, each tissue has an ECM with a unique composition and topology that is generated during

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Introduction

4

tissue development through a dynamic and reciprocal, biochemical and biophysical dialogue between the cells and the evolving microenvironment (4).

Figure 1. Composition of the ECM. The ECM is mainly composed of collagens, proteoglycans and fibronectin. Collagen triple helix results in fibers that confer tensile strength and elasticity, being the most abundant proteins of the vertebrate body. Elastin confers elasticity to the ECM. Proteoglycans, attached to long polysaccharides, regulate movement of molecules through the matrix. Fibronectin assists in the attachment of cells to the ECM. Integrins, matrix receptors located across the plasma membrane, bind to microfilaments of the cytoskeleton and translate changes in the ECM to the cell. Created with BioRender.com.

1.2 Collagens

Collagens are the most abundant proteins of the ECM. They play a fundamental role in providing the structural integrity and biomechanical properties of different tissues. In vertebrates, 28 types of collagens have been described (I-XXVIII) which are divided into several families depending on the molecular structure and assembly mode (5). The most important are the fibrillar collagens (I-III, V, XI, XXIV and XXVII) that form the backbone of the collagen fibrils bundles within the interstitial matrix surrounding cells, and basement membrane-forming collagen IV (6). Fibrillar collagens form homotrimeric (three identical α-chains) or heterotrimeric (two or three distinct polypeptide chains) molecules. Each α-chain consists of a major uninterrupted triple helical or collagenous domain -characterized by the repeating amino acid motif Gly-X-Y, where X and Y are commonly proline (Pro) and hydroxyproline (Hyp)- flanked by N- and C- terminal non- collagenous domains, i.e. the N- and C-propeptides (Figure 2).

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Introduction

5

Figure 2. Structure of human type I collagen. Type I collagen is a long ( 300nm long,  1.5 nm thick) heterotrimeric molecule composed of two 1 chains and one 2 chain. The molecule consists of three domains: the N-terminal non-triple helical domain (N-telopeptide), the central triple helical domain and the C-terminal non-triple helical domain (C-telopeptide). Created with BioRender.com.

Collagen fibrils are formed by a complex biosynthetic pathway involving numerous intracellular and extracellular steps carried out by various enzymes and chaperones, all of which contribute to the structure and biomechanical properties of the final fibrils (7). These steps include PTMs, chain association and folding, secretion, procollagen processing, self-assembly and progressive cross-linking (Figure 3).

As exemplified for human type I collagen, after synthesis on the ribosome and their import into the rough endoplasmic reticulum (RER), collagen chains are subjected to a series of PTMs resulting in the assembly of procollagen chains. These include hydroxylation of specific Pro and lysine (Lys) residues, N- and O-linked glycosylation, disulphide bonding and prolyl cis- trans isomerization. Association of the three α-chains occurs through a process governed by the C-terminus, and the formation of the triple helix is propagated towards the N-terminal end in a zipper like fashion to form the procollagen molecule. This precursor molecule is transported to the Golgi network where it is packaged into specialized secretory vesicles prior to export into the extracellular medium. Formation of fibrils from procollagen chains requires their proteolytic processing. The N- and C-propeptides are cleaved off by metalloproteinases belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein1)/Tolloid-like families respectively, yielding the tropocollagen molecule, which retains a short portion of the propeptides termed telopeptides. Then, Lys or hydroxylysine (Hyl) residues within these non-collagenous domains are oxidatively deaminated by lysyl oxidase (LOX), yielding the corresponding aldehydes which constitute the initiation products for the cross-liking formation. Within hours of helix formation, these telopeptide aldehydes spontaneously react with helical Lys or Hyl to form immature cross-links, which further react

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Introduction

6

among them and with remaining Lys or Hyl residues over months/years to form permanent cross- links

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.

Figure 3. Type I collagen biosynthesis and cross-linking. During the synthesis of the pro chains in the RER, specific PTMs take place (i.e. hydroxylation of specific Lys and Pro residues). Following these modifications, the alpha chains associate with one another and fold into a triple helical molecule to form a procollagen molecule that is secreted into the extracellular space. Outside the cell, the N- and C- propeptides are cleaved to release a collagen molecule. The collagen molecules spontaneously self-assemble into a fibril that is stabilized by intra- and inter-molecular covalent cross-linking. Created with BioRender.com.

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Introduction

7

1.2.1 Post-translational modifications of collagen

One of the characteristic features of collagen is its extensive PTMs, most of which are unique to collagen protein and have major effects on molecular assembly, stability, fibrillogenesis and cross-linking. Such modifications include hydroxylation of Pro and Lys residues, glycosylation of specific Hyl residues, oxidative deamination of Lys/Hyl in the telopeptide domains of the molecule and subsequent intra/intermolecular covalent cross-linking (10).

The conversion of specific triple-helical Pro into Hyp is essential for proper assembly and thermal stabilization of collagen triple helices through intramolecular hydrogen bonding.

Hydroxylation occurs mainly on Pro in the Y position of the Gly-X-Y triplet, through the action of prolyl 4-hydroxylases (P4H), and also to a much lesser extent on Pro in the X-position, through the action of prolyl 3-hydroxylases (P3H) (7,11).

Another critical factor that impact the structural and biomechanical properties of the collagen network are the PTMs of Lys residues. Lys modifications of collagen are highly regulated sequential processes that take place inside and outside the cell. In the cell, specific Lys residues both in the helical and telopeptide domains are hydroxylated to form Hyl. Then specific helical Hyl residues are glycosylated by the addition of galactose or glucose-galactose. Specific enzymes catalyse each one of these modifications. Outside the cell, specific residues at the telopeptides are subsequently modified by LOX.

The extent of Lys hydroxylation of collagen is highly variable in comparison to that of Pro. It differs from one genetic type to another and, even within the same genetic type I collagen, it significantly varies depending on the tissues and the tissue’s physiological/pathological conditions. Furthermore, a difference in the extent of Lys hydroxylation exists between the helical and telopeptide domains of a type I collagen molecule (12–14). This variable behavior is explained by the existence of various enzymatic systems that contribute to Lys hydroxylation of collagen. Three isoforms of lysyl hydroxylases (LH; EC 1.14.11.4), also known as procollagen- lysine, 2-oxoglutarate 5-dioxigenase (PLOD), have been described in vertebrates: LH1, LH2 and LH3. Just as the P4Hs and P3Hs, lysyl hydroxylases require Fe²⁺, 2-oxoglutarate, O2 and ascorbate for their activity. These isoforms are encoded by PLOD1, PLOD2 and PLOD3 genes, respectively (15–17). In addition, an alternatively spliced variant of LH2 (LH2b) with an additional 63bp-exon 13A has been reported

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. All of them are expressed during embryonic development in mice, but the expression levels vary between the isoforms in different developmental stages, showing that the LH genes are specifically regulated during organogenesis (19).

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Introduction

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LH1 hydroxylates mostly Lys in the helical domain of collagen I and III, especially the residues involved in cross-linking. In humans, mutations in LH1 cause Ehlers-Danlos (EDS) syndrome type VIA, a generalized connective tissue disorder characterized by hypermobile joints, hyperextensible skin and kyphoscoliosis

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. LH3 is a multifunctional enzyme that hydroxylates mostly Lys in the triple-helical region of collagen types II, IV and V and also displays galactosyltransferase and glucosyltransferase activities (22). Mutations in the LH3 gene, PLOD3, cause several congenital connective tissue malformations (23). Finally, LH2 gene expression is associated with Lys hydroxylation in the telopeptide domains of collagen I (12).

LH2b is the major form of LH2 and is mainly seen in tissues rich in fibrillar collagens. Several experimental evidences confirm that LH2b is indeed the telopeptildyl LH. Expression studies in osteoprogenitor cells reveal that the elevation of Lys hydroxylation in the telopeptides of type I collagen coincides with a higher expression of LH2 mRNA (13) Overexpression of LH2b in osteoblasts results in an increase in the hydroxyallysine (hydroxy-α-aminoadipic acid-δ- semialdehyde, Hylâld)-derived collagen cross-links, but when underexpressed, allysine (α- aminoadipic acid-δ-semialdehyde, Lysâld) and its derived cross-links are significantly increased (24). Mutations in LH2 are known to cause Bruck syndrome type 2, a rare autosomal-recessive disorder in the osteogenesis imperfecta spectrum characterized by bone fragility and congenital joint contractures and a severe reduction or elimination of telopeptide Hylâld-derived cross-links (25,26). PLOD2 expression have been described to be strongly up-regulated by hypoxia and transforming growth factor  (TGF-). Hypoxia is an important microenvironmental factor that stimulates tissue fibrosis (27,28). Hypoxia-inducible factor-1 (HIF-1) stimulates PLOD2 mRNA expression in various cell types (29). HIF-1regulated LH2 expression affects the composition and mechanical properties of human fibroblast -derived ECM (30). TGF-β is another well-known stimuli for the synthesis and deposition of a fibrotic ECM. PLOD2 expression and pyridinoline cross-links are strongly enhanced by TGF- in fibrotic pathologies (31).

1.2.2 Procollagen processing

A key step in collagen fibril formation is the removal of the globular N-and C-propeptides from the secreted procollagen molecule in the extracellular environment by the procollagen N- proteinases belonging to the ADAMTS and the procollagen C-proteinases BMP1/Tolloid-like families (32–34). Procollagen processing has a dramatic influence on fibril formation. The removal of the propeptides diminishes the solubility of the resulting tropocollagen molecule and facilitates the assembly of higher order fibrils made up of triple helical tropocollagen units organized in the characteristic quarter stagger array.

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Introduction

9

The removal of the C-propeptide is accomplished by members of the tolloid family of zinc metalloproteinases: BMP-1, mammalian tolloid (mTLD) and tolloid like 1 (TLL-1) (32–34).

This process is essential for the assembly of collagen fibers as it markedly decreases the critical concentration required for fibril assembly and triggers the self-assembly of collagen into fibrils.

When reconstituted in vitro, results in the spontaneous aggregation of triple-helical molecules into fibrils (35).

N-proteinase activity is provided by members of the ADAMTS family: ADAMTS-2, ADAMTS-3 and ADAMTS-14 (36–38). There is tissue specificity of expression of these enzymes. ADAMTS-3 is the principal cartilage and type II procollagen N-proteinase; whereas ADAMTS-2 and -14 are more important in type I collagen-rich tissues such as skin, bones, tendon and aorta. All three enzymes are able to process procollagen types I, II, and III in vitro. The critical role of ADAMTS2 in this process is illustrated in the dermatosparatic type of Ehlers-Danlos syndrome, a genetic disease caused by mutations in the Adamts2 gene. In the absence of ADAMTS2 activity, pN-procollagen (collagen still retaining its N-terminal domain) accumulates and leads to the formation of highly disorganized collagen fibers causing the extreme skin fragility that is the hallmark of the disease (39,40).

1.2.3 Collagen cross-linking

Covalent collagen cross-linking by means of LOX is the final step in the biosynthesis of collagen and is essential for the physical and mechanical properties of collagen fibrils and the stabilization of the collagen network (41). The process of cross-linking is dynamic and the pathways vary depending on the tissues and tissue’s physiological state. In this process, lysine modifications at the telopeptides play a crucial role.

The formation of these cross-links starts with the oxidative deamination of the ε-amino group of specific Lys or Hyl residues within the C- and N-terminal telopeptides by LOX, leading to the formation of the aldehydes Lys^ald and Hyl^ald. In type I collagen, there are two residues of Lys or Hyl in the C-telopeptides (both from the 1 chains) and three in the N-telopeptides that can be oxidized by LOX.

Following this reaction two related pathways of cross-linking can take place: the allysine pathway (derived from a Lys in the telopeptide) and the hydroxyallysine pathway (derived from a Hyl in the telopeptide). Subsequently, the Lys^ald or the Hyl^ald reacts either with another aldehyde in the same molecule or with a Lys, Hyl or histidine (His) residue in neighboring collagen molecules in order to form di-, tri-, or tetra- functional cross-links (Figure 4). The presence of such complex intermolecular cross-links demonstrates the highly specific molecular packing arrangement in the fibril, as the amino acid residues involved in cross-linking are relatively sparse in collagen and they need to be in correct proximity (442-44).

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Introduction

10

Figure 4. Major cross-linking pathways of collagen. The Lys in the telopeptides can be converted into Hyl by lysyl hydroxylase 2 (LH2). The Lys and Hyl in the telopeptides can be converted into the aldehydes Lysâld and Hylâld by lysyl oxidase (LOX) and/or LOX-like 1-4 (LOXL). After this reaction happens, two cross-linking pathways can take place: the allysyne (Lysâld) mainly occurs in soft tissues, whereas the hydroxyallysine (Hylâld) happens principally in skeletal tissues. Amino acids from the telopeptide and the triple helix are marked in blue and pink, respectively. Created with BioRender.com.

The extent of Lys hydroxylation in the telopeptide domain and that in the juxtaposed helical domain of a neighboring collagen molecule determine the relative abundance of these two cross-linking pathways in the different tissues. A major cross-link in soft connective tissues is derived from an intramolecular aldol condensation product (ACP) formed between two Lysâldin the N-telopeptide domain. This ACP matures into a tetravalent intermolecular cross-link, dehydrohistidinohydroxymerodesmosine (deH-HHMD), by reacting with His and Hyl residues in the triple helix on a neighboring molecule (45). Collagen deposited in skin and cornea is also mainly cross-linked via the Lysâld pathway with almost no Hylâld cross-links present (46). In these tissues, Lysâld in the C-telopeptide domain reacts with specific Lys or Hyl in the triple helix that results in the divalent cross-link dehydro-lysinonorleucine (deH-LNL) and dehydro- hydroxylysinonorleucine (deH-HLNL), respectively. deH-HLNL can pair with a helical His to form the trivalent cross-link histidinohydroxylysi- nonorleucine (HHL) (47).

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11

However, very recently this cross-link has been proposed to be an artifact (48). By contrast, in tissues that support higher mechanical strength such as bone, cartilage, tendon, dentin, aorta and ligaments, collagen cross-links are mainly derived from the Hylâld pathway (49). Hylâldin the telopeptide can react with a helical Lys or Hyl resulting in the divalent iminium cross-links deH- HLNL and dehydro-dihydroxylysinonorleucine (deH-DHLNL). These cross-links undergo a spontaneous Amadori rearrangement leading to more stable divalent keto-imines that react with either a Lysâld or a Hylâld from the telopeptides, resulting in the trivalent pyrollic cross-links (d- PRL, deoxypyrrole; PRL, pyrrole) or trivalent pyridinoline cross-links (HP, hydroxylysylpyridinoline; LP, lysylpyridinoline) (44).

1.3 Lysyl oxidases

Collagen cross-linking plays a critical role in the stiffness and biomechanical properties of the ECM and thereby in normal tissue integrity. Cross-linking between collagen molecules occurs in both enzymatic (regulated) and non-enzymatic (non-regulated) manners. Enzymatic collagen cross-linking is mainly mediated by members of the lysyl oxidase (LOX; EC 1.4.3.13) family of secreted amine oxidases (50). The lysyl oxidase family are copper-dependent amine oxidases that oxidize primary amine substrates to reactive aldehydes. The best studied role of the LOX enzymes is their capacity to oxidize Lys residues in elastin and Lys and Hyl residues in collagen to initiate their covalent cross-linking. These enzymes have biological functions that extend beyond this fundamental biosynthetic role, with contributions to regulation of gene transcription, development, angiogenesis, cell proliferation and differentiation and tissue and repair remodeling

(51–53)

. The up-regulation of LOX isoforms is associated with the imbalance between ECM degradation and synthesis involved in fibrotic disorders including liver fibrosis, glaucoma, cardiac fibrosis, diabetic nephropathy, atherosclerosis and pulmonary fibrosis

(54–

56)

.

1.3.1 Domain organization of lysyl oxidases

Five different LOX enzymes have been identified in mammals, the prototypical lysyl oxidase (LOX) and four lysyl oxidase like-1 through lysyl oxidase like-4 (LOXL1 - LOXL4) (Figure 5).

While being the products of distinct genes, all five members share amine oxidase function mediated by a common catalytic domain, that is highly conserved among species, including fly, mouse, rat, chicken, fish and human (57). This C-terminal domain contains a copper-binding motif, a cytokine receptor-like (CRL) domain, a lysine tyrosylquinone (LTQ) cofactor-moiety, and twelve cysteine residues (58). Both copper and LTQ cofactor are required for the oxidase activity. The copper ion is coordinated in LOX by three histidine residues (H292, H294 and H296).

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12

These residues are essential to the formation of LTQ, which requires copper and oxygen and is essential for LOX and LOXL1 enzymatic activity

(59)

. The His 303 has been identified as a key residue that acts as the catalytic base for the LOX enzyme to function properly (60,61).

LOX family oxidases differ significantly at their N-terminal domains, which are proposed to provide the enzymes with specific capabilities. Although all members have a signal peptide and are secreted to the extracellular environment, LOX and LOXL1 contain a distinctive N-terminal propeptide, whereas the N-terminus of LOXL2, LOXL3 and LOXL4 contains four scavenger receptor cysteine-rich (SRCR) domains.

Figure 5. Domain organization of members of the lysyl oxidase (LOX) family. LOX proteins share a highly conserved catalytic carboxy terminal domain that contains a copper-binding motif and a lysyl tyrosyl quinone (LTQ) cofactor. The amino terminal ends are more divergent, with LOX and LOXL1 containing pro-sequences (including a proline-rich domain in LOXL1), and four scavenger receptor cysteine-rich (SRCR) in LOXL2-4, describe to participate in protein conformation, cell adhesion and protein-protein interactions. The proteolytic processing sites in LOX and LOXL2 are indicated in red. Created with BioRender.com.

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The SRCR domains were likely acquired with the appearance of ECM in early metazoans (62). These domains are frequently found in cell surface proteins associated with the immune system, and are suggested to be involved in protein-protein interactions in the ECM (63).

1.3.2 Pathophysiological functions of lysyl oxidases 1.3.2.1 Lysyl oxidase (LOX)

LOX was the first isoform to be cloned and characterized and is the best studied to date

(64)

. In humans, LOX gene is localized on chromosome 5 and codifies a 417 amino-acid protein that is the most abundant expressed isoform in skeletal muscle, heart, kidneys and lungs. As aforementioned, LOX is required for the biosynthesis of normal collagens and elastin, and therefore is critical for vascular, mineralized and non-mineralized connective tissues. Consistent with this role, the disruption of LOX gene is associated with profound abnormalities as clearly demonstrated in a LOX-deficient mouse model. Lox-null mice are perinatal lethal with cardiovascular fragility, burst arterial aneurysms, ruptured diaphragm and fragmented elastic fibers, suggesting that LOX has an essential role in the development and function of the cardiovascular system (65,66). The Lox-null mice also display impaired airway development in the lungs and abnormal collagen and elastin fibers in the skin (67).

LOX-mediated matrix remodeling provides strength and resistance against mechanical stress, an essential feature in the vasculature. Recently, mutations in the LOX gen has been associated with the development of aortic aneurysms and dissections in humans (68,69). Elevated expression of LOX and LOXL1 enzymes seems to be a protective factor against aneurysmal progression in patients with Marfan syndrome, a hereditary condition characterized by an aberrant formation of elastic fibers due to mutations in the gene encoding fibrillin-1 (70) . LOX also plays an important role in heart damage. Injury to the myocardium results in a fibrotic response with enhance ECM synthesis and deposition. The increased in chamber stiffness observed during cardiac remodeling of the damage heart is associated with an enhanced expression and/or activity of LOX and LOXL2 and therefore augmented collagen cross-linking (71,72).

LOX also plays a fundamental in the formation of bone and cartilage, where the biomechanical properties of the collagen matrix are determined by the quantity and quality of LOX- mediated cross-links. Ehlers-Danlos (EDS) syndrome VII A and B belongs to a group of connective tissue disorders characterized by severe joint hypermobility and hip dislocation and involve skipping of exon 6 from either COL1A1 or COL1A2 (73). The resulting mutant collagen molecule losses the N-proteinase cleavage site and the N-telopeptide cross-linking Lys.

Consequently, the cross-linking is defective and severely impairs fibril formation. Another genetic bone disorder linked with defective LOX-mediated collagen cross-linking is Bruck syndrome. Mutations in PLOD2 leads to diminished Hyl^ald, impeding proper formation of bone collagen (74).

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14

The effects of LOX on cell biology and physiology extend beyond its role in cross-linking having physiologic substrates in addition to collagens and elastin. It also acts on few soluble substrates such as fibroblast growth factor-2 (FGF-2) and TGF- inhibiting their signaling (75,76). LOX activity is required for the optimal response of platelet-derived growth factor (PDGF) receptor to PDFG-BB ligand in smooth muscle cells and megakaryocytes (77,78). LOX can also be internalized by fibroblasts and interactions with intracellular proteins such as histone H1 has also been described (79). Furthermore, the nuclear expression of LOX is an independent prognostic factor in rectal cancer patients (80).

1.3.2.2 Lysyl oxidase like-1 (LOXL1)

LOXL1 isoform is structurally and evolutionary related to that of LOX isoform. The LOXL1 gene located on chromosome 15 encodes a 574 amino acid protein predominant in the pancreas, skeletal muscle, spleen, heart and lungs. Loxl1-null mice experience skin, uterine and lung abnormalities and females exhibit uterine prolapse (81–83). Based on these observations LOXL1 has been linked primarily to elastin maturation. This isoform not only catalyzes the cross- linking reaction but also contributes to the scaffolding leading to elastic fiber assembly.

Moreover, a deficiency in trabecular bone in both long bones and vertebrae has been observed in female mutant mice, suggesting that LOXL1 also contributes to collagen maturation and that sex hormone regulation of LOXL1 may be important for its biological control (84).

Several studies associate an aberrant regulation of expression of LOXL1 and/ or missense mutations combined with environmental stressors with pseudoexfoliation syndrome (XSF). XSF is a complex systemic disorder characterized by the aberrant deposition of fibrillar material predominantly in the eye and visceral organs and is the most identifiable cause of glaucoma (85,86). Additionally, this isoform has been identified as a prominent component of the fibrillar XFS deposits along with other elastic fiber components in intra- and extraocular locations, being this observation consistent with the role of LOXL1 in elastic fiber formation (87,88).

LOXL2 is encoded on chromosome 8, includes 774 amino acids and is principally expressed in testis, ovary, thymus, skin and lung cells. LOXL2 plays an important role in the stabilization of basement membrane networks by cross-linking of collagen IV in the kidney glomerulus and vasculature (89,90). Multiple studies have reported the implication of LOXL2 in a wide spectrum of biological processes, including gene transcription, cell, migration, adhesion and differentiation

(91,92)

. Loxl2-null and overexpressing mice have recently been generated (93). LOXL2 deletion results in perinatal lethality in 50% of the mice associated to severe heart defects. Homozygous mice overexpressing LOXL2 do not exhibit any particular defect, but most of the male are sterile with poor testicle formation and low sperm production due to epithelial disorganization, fibrosis and inflammation.

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Introduction

15

It is noteworthy that in a model of squamous cell carcinoma LOXL2 overexpression is associated with accelerated tumor progression and severity compare to that of the Loxl2-null mice, which develop less tumors and of smaller size (94). These actions seem to occur through an inhibition of the Notch pathway mediated by LOXL2 binding to the Notch promoter, thus indicating a fundamental role of LOXL2 in tumor initiation and progression.

LOXL3 is encoded on chromosome 2 and is composed of 753 amino acids. It is predominant in ovary, uterus, testis, heart, pancreas and skeletal muscle. Loxl3-null mice have recently been characterized and are perinatal lethal, exhibit cleft palate and vertebral defects (95).

Loxl3-null embryos show less collagen cross-links and impairment in lung development accompanied by deformed and smaller thoracic cavities (96). Recently, an autosomal recessive mutation in the LOXL3 gene has been described to cause Stickler syndrome, a collagenopathy mostly involving cartilage-specific type II and XI collagens, characterized by cleft palate, ocular abnormalities hearing loss and joint problems (97). These observations suggest that LOXL3 isoform is mainly involved in the cross-linking of cartilage collagen.

LOXL4 is the latest reported member of the LOX family. LOXL4 gene is located on chromosome 10 and codifies a 756 amino acid protein (98). Relatively low levels of LOXL4 mRNA have been detected in multiple human tissues, with prevalence in placenta, lung, kidney, pancreas, testis and ovary. Recent studies demonstrate the implication of this isoform in the remodeling of the vascular ECM, where its expression is strongly regulated by TGF-. It is also involved in the process of corneal wound healing, where LOXL4 is transcriptionally regulated by retinoic acids (99,100). LOXL4 overexpression is associated with certain tumors and carcinoma cell lines. Besides, shorter splice variants of LOXL4 has been propose to stimulate metastasis whereas full length LOXL4 may have tumor inhibitory properties (101). No phenotype of the knockout mouse model has been reported yet.

LOXL4 is the latest reported member of the LOX family. LOXL4 gene is located on chromosome 10 and codifies a 756 amino acid protein (98). Relatively low levels of LOXL4 mRNA have been detected in multiple human tissues, with prevalence in placenta, lung, kidney, pancreas, testis and ovary. Recent studies demonstrate the implication of this isoform in the remodeling of the vascular ECM, where its expression is strongly regulated by TGF-. It is also involved in the process of corneal wound healing, where LOXL4 is transcriptionally regulated by retinoic acids (99,100). LOXL4 overexpression is associated with certain tumors and carcinoma cell lines. Besides, shorter splice variants of LOXL4 has been propose to stimulate metastasis

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16

whereas full length LOXL4 may have tumor inhibitory properties (101). No phenotype of the knockout mouse model has been reported yet.

1.3.3 Regulation of lysyl oxidases

In order to achieve their critical functions in ECM homeostasis a precise regulation of the expression and activity of this family of enzymes is required. Aberrant expression of these enzymes and/or dysregulated oxidase activity are responsible for many pathological conditions, such as tissue fibrosis or cancer. LOX enzymes are temporal-spatially regulated at both transcriptional and post-transcriptional levels.

TGF-, a central mediator in the regulation of ECM remodeling, has been show to upregulate LOX mRNA levels through integrated Smad3, PI-3 kinase and MAPK signaling in a dose- and time-dependent manner (102). Also, the pro-inflammatory cytokine TNF- stimulates LOX expression, which may play an important role in progressive cardiac fibrosis

(103)

. The forkhead box M1b (FoxM1b) transcription factor, overexpressed in human cancers and correlated with poor prognosis, directly binds to the promoters of LOX and LOXL2 genes, inducing their expression and activating the Akt-Snail pathway which drives epithelial-mesenchymal transition (EMT), hepatic fibrosis and metastasis of hepatocellular carcinoma

(104)

. On the contrary, GATA-3 transcription factor negatively regulates LOX expression through LOX promoter methylation

(105)

. Hypoxia is an important factor implicated in the development of many diseases, including many types of fibrosis and tumorogenic processes. LOX mRNA levels are highly up-regulated by HIF-1 at the transcriptional level

(106)

. LOX is also highly responsive to HIF-2, which is mediated by two functional hypoxia inducible response elements recently identified in the LOX promoter

(107)

.

At the post-transcriptional level, the interaction of LOX with other ECM components exerts regulatory roles in determining the spatial distribution, substrate specificity, oxidase activity and their physiological and pathological functions. Indeed, the increased LOX enzyme activity upon TGF- stimulation is delayed and of lower magnitude than the increase in its mRNA, suggesting rate-limiting post-translational regulation of LOX.

In the particular case of LOX and LOXL1, these isoforms are secreted as proenzymes and the cleavage of the propeptide is required for their activation

(108,109)

. Post-translational processing of immature LOX proenzyme involves removal of the signal peptide by cleavage at Cys21-Ala22 and N-terminal glycosylation in the ER and Golgi apparatus. Then the 50 kDa precursor is secreted to the extracellular space, where it is cleaved between Gly168 and Asp169 by BMP1-related metalloproteinases (Figure 5), including BMP1, mTLD and mTLL-1 and -2.

This proteolytic processing releases the mature enzymatically active form of LOX of 32 kDa and an 18 kDa bioactive N-terminal propeptide fragment. Considering the capacity of LOX to cross-

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Introduction

17

link fibrillar collagen, it is remarkable that the same family of enzymes responsible for the processing of collagen is also involved in the proteolytic activation of LOX. Several other ECM proteins regulate the proteolytic activation of the LOX precursor (proLOX). Fibronectin interacts with proLOX and its critical for its proteolytic activation, which is decreased in fibronectin-null mouse embryonic fibroblasts

(110)

. Fibulin-4 stimulates the processing of the precursor, as the proteolytic activation of LOX is reduced in fibulin-4 deficient osteoblasts but is rescued by the addition of recombinant fibulin-4

(111)

. Besides, thrombospondin-1 binds to the helical cross- linking site of the collagen molecule inhibiting the proteolytic processing of LOX by BMP1

(112)

. However, several experimental evidences reveal the existence of different mature forms that coexist and contribute to LOX-mediated actions

(112–114)

. To this respect, using a large scale and non-biased approach, LOX has been recently identified as a potential substrate of the procollagen N-proteinase ADAMTS14

(115)

.

Many evidences indicate that LOXL1 isoform is also subject of a proteolytic event to yield the active form

(108,116)

. Nevertheless, the proteases potentially involved, the specific cleavage site on the protein sequence and the effect of this cleavage on LOXL1 function remains to be identified.

LOXL2 is also processed in the extracellular space. Proprotein convertase subtilisin/kexin type 6 (PACE4) is the major protease that process this isoform between Lys 317 and Ala 318, generating a 65 KDa fragment that lacks the first two SRCR domains (Figure 5).

The proteolytic processing of LOXL2 is required for cross-linking of collagen IV in the ECM although it does not affect the oxidation of the small, soluble substrate 1,5-diaminopentane in vitro. Moreover, this processing is not essential for the amine oxidase activity of LOXL2 in vitro with collagen IV or tropoelastin as substrates (117,118).

1.3.4 Lysyl oxidases as pharmacological targets in fibrotic diseases

Numerous studies have begun to address the potential value of inhibiting LOX family of enzymes and thereby decreasing the amount of insoluble cross-linked collagen as a therapeutic approach to treat fibrotic diseases. This concept has been largely demonstrated in animal models of fibrosis. For instance, LOX inhibition or knockdown of LOX expression alleviates the lung fibrosis in a bleomycin-induced model of lung fibrosis

(119)

. Also, the fibrotic response in the liver of mice treated with carbon tetrachloride (CCI4) is reverted upon LOX inhibition

(120)

.

-aminopropionitrile (BAPN) is a well-known specific and irreversible inhibitor of LOX activity.

This compound exerts its inhibitory actions by direct binding to the catalytic site of LOX or LOXL isozymes where it forms an irreversible covalent bond, possibly involving the primary amine of BAPN and carbonyl group in the LOX catalytic site, blocking the conversion of Lys to Lys^ald residues in substrates proteins

(121)

. However, conflicting results have been reported for

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Introduction

18

the inhibition of LOX and LOXL2

(122)

. Its effects on LOX activity and cross-link formation have been evaluated in numerous studies. BAPN prevents the increase in matrix metalloproteinases (MMPs), collagen cross-linking and improves cardiac function in volume- overloaded rats

(123)

. It slows down angiogenesis and migration of human endothelial cell in vitro

(124)

, and reduces body weight gain and improves the metabolic profile in diet-induce obesity in rats where LOX is overexpressed

(125)

. Nonetheless, its pharmacological use as a systemic drug is not indicated. It has been observed that prolonged administration of BAPN evokes a weak neurotoxicity and reduces the mechanical strength of bones

(126,127)

. Further, lathyrism, a disease characterized by extensive disruption of connective tissue which severely disturbs bone mechanical strength and blood vessel mechanics, is caused by the

excessive and long-term consumption of Lathyrus odoratus (sweet pea) seeds, which contain high amounts of BAPN and related compounds (128).

Given these constraints, alternative approaches have been assessed. Monoclonal antibodies against LOX and LOXL2 have demonstrated their ability to ameliorate cardiac dysfunction and fibrosis in response to pressure overload or myocardial infarction in mice

(129)

. AB0023, a specific noncompetitive allosteric inhibitory monoclonal antibody, prevents fibroblast activation, reduces tumor formation, prevents pathological fibrosis across several disease models and reverses bleomycin-induced lung fibrosis

(130)

. However, its humanized variant Simtuzumab (GS 6624) has been shown to be ineffective in patients with bridging fibrosis or compensated cirrhosis caused of no significant clinical benefit in reducing liver and lung fibrosis caused by nonalcoholic steatohepatitis and in patients with primary sclerosing cholangitis

(131,132)

. However, and despite promising preclinical data, this drug showed no significant clinical benefit in reducing liver and lung fibrosis

(133)

.

Besides monoclonal antibodies, several other small molecules that directly inhibits catalytic activity of LOX and LOXLs have been developed for the treatment of fibrosis and cancer. To illustrate, CCT365623 is an orally bioavailable LOX inhibitor with antimetastatic properties

(134)

. PXS-5153A, a novel dual LOXL2/LOXL3 inhibitor, reduces collagen cross- links and disease severity in two liver fibrosis models

(135)

. Alternative approaches derived from the use of metal chelators, which reduce copper availability and therefore impair LTQ biogenesis.

achieve an effective reduction of LOX activity. Cancer patients treated with tetrathiomolybdate (TM), a copper-chelating agent that prevents the cellular uptake of copper showed disease stabilization for several months, or even regression

(136)

.

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Introduction

19 1.4 ECM remodeling and fibrosis

The precise organization and orientation of ECM components creates a highly organized topology that contributes to the functional properties of the matrix. This topology is determined by a continuous dynamic remodeling not only in homeostasis but also in response to damage or disease. ECM remodeling is achieved by a careful balance between matrix synthesis, secretion, modification and enzymatic degradation. Changes in matrix homeostasis affect not only the biomechanical properties of the matrix but also the resulting biophysical properties, both of which are crucial for normal development and normal tissue function. For instance, changes in the synthesis or degradation of one or more ECM components, or alterations in ECM structure due to modifications in the expression or activity of ECM remodeling enzymes can lead to life- threatening pathological conditions such as fibrotic diseases and cancer

(1–3)

.

ECM remodeling is essential for normal organ homeostasis, wound healing and development but excessive deposition can lead to organ dysfunction that results in fibrotic and degenerative diseases. Fibrosis is the excessive accumulation of ECM that can lead to distortion of tissue architecture and loss of organ function. This pathology commonly results from a wound healing response to repeated or chronic injury or tissue damage. A broad range of prevalent chronic diseases can give rise to fibrosis, including diabetes, hypertension, viral and non-viral hepatitis, heart failure and cardiomyopathy, idiopathic pulmonary disease, scleroderma, and cancer. Fibrosis resulting from these and other diseases can lead to failure of liver, lung, kidney, heart, or other vital organs as excessive ECM replaces and disrupts parenchymal tissue

(137,138)

. Consequently, severe fibrosis is estimated to account for up to 45% of all deaths in the developed world. Current therapies for fibrosis are few and of limited efficacy. Therefore, there is an urgent need to understand how fibrosis may regress and to identify potential therapeutic approaches (5).

Type I collagen is the mayor constituent of the fibrotic ECM. However, it is not just the quantity of collagen that defines fibrosis. The quality of collagen as determine by its post- translational modifications, actively drives disease progression. A marked modification is the increased cross- linking mediated by LOX, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in Hylâld-derived cross-links at the expense of Lysâld cross-links. This results in irreversible fibrosis, as collagen cross-linked by Hylâld shows a high resistance toward MMPs. The family of LH contributes to the conversion of Lys into Hyl in the telopeptides of the collagen molecule, leading to the formation of Hylâld cross- links by LOX. Therefore, fibrotic conditions are linked to increased expression and/or activity of LOX and LH enzymes

(139–142)

.

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20

1.5 Cell-derived ECM-based biomaterials for tissue engineering

Tissue engineering is a developing field that aims to combine cells, scaffolds, and growth factors to build artificial substitutes with biological function in vitro. These constructions can be used to repair tissue defects and replace the loss of function or failure of the organization of damaged tissues and organs. Scaffolds provide a suitable microenvironment to sustain the growth of seeded cells and cytokines; they supply mechanical support for tissue defects and show good histocompatibility when implanted. Consequently, there is a great interest in the development of suitable scaffold materials in the field of tissue engineering research

(143–145)

.

Many natural and synthetic scaffold materials have been developed and tested for the repair and restoration of numerous tissues. Among these, biological scaffolds derived from cell and tissue-derived extracellular matrix (ECM) have shown great promise in tissue engineering given the critical role of the ECM for maintaining the biological and biomechanical properties, structure, and function of native tissues. Cell-derived ECM can be obtained from autologous cells cultured under sterile conditions in vitro, thereby avoiding the shortcomings of decellularized ECM derived from tissue (i.e. potential pathogen transfer, inflammatory or anti-host immune responses, uncontrollable degradation). Besides, cell-derived ECM scaffolds are more readily customizable through the use of different cell types, as opposed to tissue-derived ECM scaffolds, which require patient-specific cells. These cell-derived ECM also provide the appropriate microenvironment to promote cell proliferation, adhesion, and differentiation of cells, as they maintain the desired biological elasticity, topology and biomechanical characteristics, as well as speed up the repair of damage tissues

(145–147)

. Therefore, there remains a strong interest in developing techniques and protocols that boost the innate capacity of cells to create their own ECM in vitro.

The construction of stable engineered cell-derived ECM depends of the rapid and efficient deposition of ECM components. Nevertheless, the deposition of ECM in cell culture systems in vitro is very slow, due to the notably dilute and far from physiological extracellular medium. In the case of type I collagen, the main structural ECM molecule, a deficient PTM at the level of the cleavage of the C-propeptide, catalyzed by BMP1, and the formation of covalent cross-links, initiated by LOX, has been propose to severely limit its deposition onto a stable insoluble matrix

(148,149)

. In the very dilute in vitro microenvironment, BMP1 will be deactivated before it can cleave the C-propeptide or activate the LOX precursor. Only after prolonged culture time, the cells will self-crowd the media and collagen deposition will be achieved. Several approaches have been developed to enhance this deposition in standard cell culture conditions, including ascorbic acid and serum supplementation, or the addition of inert macromolecules in the culture media in order to recreate a dense extracellular space (Figure 6)

(150,151)

. However, despite significant progress, optimal conditions for rapid and efficient deposition of collagen are still missing.

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Introduction

21

Figure 6. Modulation of the in vitro microenvironment using macromolecular crowding to imitate the in vivo dense extracellular space. The deposition of collagen type I in the current standard culturing systems is very slow. The addition of inert macromolecules such as dextran sulfate (DxS) or Ficoll^TM has been reported to enhance the capacity of different cell cultures to deposit abundant ECM. These molecules create more effective volume occupancy in the culture media. Consequently, they increase the relative density of procollagen and proteinases in the culture media. This facilitates the cleavage of the propeptides by their respective proetienases and speed up the formation of collagen. Created with BioRender.com.

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Aims and objectives

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Aims and objectives

25 2. Aims and objectives

The present doctoral thesis has been focused on the study of the regulation of lysyl hydroxylase 2 (LH2) and lysyl oxidase (LOX) enzymes, which are critical for the biomechanical functions and deposition of fibrillar type I collagen onto the ECM, and its application in tissue engineering.

Specific objectives of this thesis are:

1. To analyze the signaling pathways regulating LH2 expression as a way to understand the molecular mechanisms contributing to tissue stiffness and pathological fibrosis.

i. To identify of the molecular determinants underlying hypoxia and TFG--induced PLOD2 gene expression.

ii. To study the impact of PLOD2 expression driven by hypoxia and TGF- on collagen biosynthesis and deposition onto the ECM in vitro.

2. To analyze novel molecular mechanisms regulating the biological activity of LOX isoform on collagen.

i. To demonstrate the proteolytic processing of LOX by the procollagen N-proteinases belonging to the ADAMTS (a disintegrin and metalloproteinase with trombospondin motifs) family in vitro, as well as to define their specific cleavage sites.

ii. To evaluate the contribution of post-translational modifications (PTMs) to the regulation of LOX biological activity.

iii. To study the biological effect of these mechanisms of regulation on LOX enzymatic activity in vitro.

3. To develop a protocol to enhance the capacity of in vitro cell cultures to deposit collagen onto the ECM.

i. To generate stable HEK293-based cell systems overexpressing LOX and BMP1 to recapitulate the proteolytic activation of LOX in vitro.

ii. To implement fibroblast cultures with LOX and BMP1-enriched supernatants to increase collagen deposition and generate cell-derived ECMs.

iii. To test the capacity of these fibroblast-derived matrices to regulate the differentiation of human mesenchymal stem cells (hMSCs) to adipogenic and osteogenic lineages.

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Contribution of the lysyl hydroxylase 2 and lysyl oxidase enzymes to the remodeling of the extracellular matrix. Implications in the biosynthesis of fibrillar collagen and its application in tissue engineering

UNIVERSIDAD AUTÓNOMA DE MADRID

PROGRAMA DE DOCTORADO EN BIOCIENCIAS MOLECULARES