A better understanding of these structural changes that occur before Hypertension (HTN) could ultimately result in a treatment that can prevent or reverse this disease state before its onset. T cells have been shown essential for the development of HTN. The aim of these murine studies was to investigate the role of the T-helper CD4⁺ lymphocytes in initiating vascular remodeling and HTN in the absence of an increased mechanical load and to investigate the role of T-helper 17 (Th17) CD4⁺ lymphocyte initiating vascular remodeling and HTN by stimulating Lysyl Oxidase (LOX). LOX is known to cross-link collagen and elastin and. Excess synthesis of collagen and elastin results in a stiffer artery and hypertension. We established L-NAME-induce HTN in wild type (WT) mice. CD4⁺ splenic lymphocytes were isolated from these mice and adoptively transferred into naïve syngeneic severe combined immunodeficient (SCID) mice. The SCID mice receiving these cells became hypertensive. Cytokine analysis demonstrated an increase in both Th1 and Th17 cytokine in HTN donor mice and of lymphocytes in the aortic infiltrates of the HTN recipient mice. The increased collagen and LOX expression in recipient mice suggest that the adoptively transfer CD4⁺ lymphocytes are associated with vascular extracellular matrix remodeling. Furthermore we examined the role of Th17 lymphocyte in aortic LOX regulation in Angiotensin II-induced hypertension. The Increase in blood pressure and Velocity Time Integral (VTI) was measured in WT Angiotensin II treated mice whereas no change was detected in the Th17 deficient (RORT KO) Angiotensin II treated group. When compared to the control group the WT group infused with Angiotensin II had higher LOX protein expression, LOX fluorescent Immunohistochemical stain and LOX activity. This group also had increased hydroxyproline levels, collagen stain, hyperplasia and aortic thickening. In contrast, the Th17 deficient mice Angiotensin II treated group had no changes in these parameters. The results provide evidence that IL-17 mediates Angiotensin II-induced hypertension and vascular dysfunction by the overstimulation of LOX. Potentially targeting T17 cells will allow for a drug-specific therapeutic approach and delay the progression of hypertension pathology.

Several members of the lysyl oxidase family of copper-dependent amine oxidases have been implicated in tumor development. The Iysyl oxidase (LOX) and LOX-like 2 (LOXL2) genes have been mapped to chromosomal regions affected by loss of heterozygosity (LOH) in several cancers, including those of the colon and esophagus. Indeed, there have been numerous reports of reduced LOX and a few reports of reduced LOXL2 expression in various cancers. Identification of microsatellite markers within the LOX locus and the LOXL2 gene allowed for evaluation ofthe status of these gene alleles in colon and esophageal tumors. There was significant LOH of the LOX locus in colon tumors that was accompanied by reduced mRNA expression and a spectrum of alterations and mutations affecting the LOX gene. This study demonstrated, for the first time, that genetic events, namely LOH, deletions and mutations ofthe LOX gene, were responsible, at least partly, for the reduction of LOX gene expression. There was also significant LOH of the LOXL2 gene in both colon and esophageal tumors. However, instead of a reduction of LOXL2 expression, there was increased expression that correlated with less differentiated tumors and absent elastosis, both indicators of poor prognosis. Further studies indicated that both LOX and LOXL2 are absent in non-invasive tumor cell lines but re-expressed in invasive cell lines, likely as part of the thelial-mesenchymal transition that occurs in the last steps of tumorigenesis to facilitate metastasis. The results presented and research strategy outlined in this dissertation will define the importance of LOXL2 amine oxidase activity and protein interactions in the critical but poorly understood process oftumor cell migration and invasion.

Metastasis is a major cause of cancer-related deaths and there is a great need for therapies aimed at preventing the spread of cancer, for which lysyl oxidase (LOX), an amino oxidase, plays a crucial role. The general aim of the thesis project was to screen and validate compounds with LOX inhibitory activities. To achieve this goal, we built a pharmacophore model of LOX inhibitors (LOXi) and screened a database of over 4400 compounds with known anti-tumoral activity in cell assays, the Biolab’s database. Top hits from the pharmacophore screening were docked to a LOX crystal structure to assess interactions between predicted inhibitors and the enzyme, and the methodology was implemented using MOE (Molecular Operating Environment) software. The pharmacophore model here developed had 5 features and produced an enrichment factor of 20 in our refinement stage. Biolab’s database screening resulted in the discovery of 344 hits. We docked the top 100 hits against a crystal structure of LOX and the results from the top 10 compounds, ranked by their interaction energy, were further analyzed. Using computer simulation software like MOE, increases the chances of finding a true inhibitor of LOX as compared to random testing of potential inhibitors, thus saving time and resources. / Metastaser är en viktig orsak till cancerrelaterade dödsfall och det finns ett stort behov av behandlingar som syftar till att förhindra spridning av cancer, där lysyloxidas (LOX), ett aminooxidas, spelar en avgörande roll. Det allmänna syftet med avhandlingsprojektet var att screena och validera föreningar med LOX-hämmande aktiviteter genom att konstruera en farmakofilmodell av LOX-hämmare (LOXi) för att screena en databas med föreningar med känd anti-tumoral aktivitet, Biolabs databas. De bästa träffarna från farmakoforscreeningen dockades till en LOX-kristallstruktur för att bedöma interaktioner mellan förutsedda hämmare och enzymet. Metoden implementerades med hjälp av programvaran MOE (Molecular Operating Environment). Den farmakospormodell som utvecklades här hade 5 funktioner och gav en anrikningsfaktor på 20 i vårt förfiningssteg. Biolabs databasscreening resulterade i upptäckten av 344 träffar. Vi dockade de 100 bästa träffarna mot en kristallstruktur av LOX och resultaten från de 10 bästa föreningarna, rangordnade efter deras interaktionsenergi, analyserades ytterligare. Genom att använda datorsimuleringsprogram som MOE ökar chansen att hitta en verklig hämmare av LOX jämfört med slumpmässiga tester av potentiella hämmare, vilket sparar tid och resurser.

L’angiogenèses par bourgeonnement est associée à une réorganisation majeure de la matrice extracellulaire (MEC). Nous avons déjà démontré que la lysyl oxydase-like 2 (LOXL2), une enzyme responsable du crosslinking de la MEC, régule la formation de vaisseaux intersomitiques dans les embryons de poisson zèbre et de capillaires en hydrogels 3D. Dans ce manuscrit, nous avons examiné les mécanismes impliqués dans cette régulation. Nous avons constaté l’association intracellulaire de LOXL2 avec la fibronectine et le collagène IV, avant d’être incorporée dans des structures fibrillaires dès l’exocytose. De plus, l’inhibition de l’expression de LOXL2 entraine des défauts de déposition de la MEC et diminue sa rigidité, inhibant secondairement la maturation des structures d'adhésion cellulaire. Alors que LOXL2 n‘est pas nécessaire pour la formation de capillaires dans un modèle 2D sur MEC de fibroblastes, les défauts de déposition de MEC sont corrélés à l'inhibition de formation des capillaires en hydrogel 3D. Ni l’addition de LOXL2 exogène, ni l’augmentation de la rigidité des hydrogels ne compense la perte d’expression de LOXL2. Enfin, nous avons pu montrer que ni l'activité catalytique ni le domaine catalytique de LOXL2 ne sont essentiels pour la formation de capillaire dans le poisson zèbre et dans les hydrogels et pour l’assemblage du collagène IV par des cellules endothéliales. L’ensemble de ces données suggère donc que les domaines SRCR de LOXL2 exprimés par des cellules endothéliales régulent l’échafaudage de fibronectine et de collagène IV dans la MEC qui est nécessaire à la formation de capillaires. / Sprouting angiogenesis is associated with major extracellular matrix (ECM) remodelling, consisting in both degradation of the microenvironment and generation of a new basement membrane. We have previously reported that lysyl oxidase-like 2 (LOXL2), an enzyme responsible for ECM crosslinking, regulates formation of intersomitic vessels (ISV) of zebrafish embryos and of capillaries in 3D hydrogels. In this manuscript we investigated the mechanisms involved. We found that LOXL2 associates with fibronectin and collagen IV intracellulary before direct incorporation in fibrillar structures of the ECM upon exocytosis. In addition, silencing LOXL2 demonstrated its involvement in ECM deposition as both composition and stiffness of the ECM were affected, which subsequently altered maturation of cell adhesion structures. Whereas LOXL2 is not required for formation of capillaries on top of a fibroblast monolayer, in a 2D assay, ECM defaults were associated with altered formation of capillaries in 3D hydrogels.Neither addition of exogenous LOXL2, nor increasing the stiffness of hydrogels could restore capillary formation. Moreover, we could show that neither the catalytic activity nor the catalytic domain were required for capillary formation in vivo and in 3D hydrogels, and for collagen IV deposition by endothelial cells. Altogether, these data suggest that the SRCR domains of LOXL2 expressed by endothelial cells regulate 3D capillary morphogenesis through scaffolding of fibronectin and collagen IV in the ECM.

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

La lysyl oxydase (LOX) est une enzyme extracellulaire dont le rôle canonique est de catalyser la réticulation des fibres de collagènes et de l’élastine, assurant ainsi l’intégrité des tissus conjonctifs. La LOX agit également dans plusieurs types cellulaires comme régulateur de différents processus biologiques, soulignant des fonctions à la fois extra et intracellulaires. Ces travaux de thèse ont contribué à améliorer la compréhension du rôle de LOX dans l’épiderme, et plus précisément au cours de la différenciation des kératinocytes. Le développement d’un modèle de culture en monocouche à confluence, et d’un modèle tridimensionnel d’épiderme reconstruit ont permis d’aborder l’étude de LOX au cours de l’induction du programme de différenciation des kératinocytes et du processus de différenciation terminale conduisant à la formation d’un épiderme pluristratifié, cornifié et fonctionnel. L’expression de LOX est induite au cours des premières étapes de la différenciation de kératinocytes primaires ainsi que d’une lignée de kératinocytes immortalisés. Grâce à l’établissement de lignées de kératinocytes éteignant l’expression de LOX de façon stable, nous avons mis en évidence l’implication de la protéine LOX, indépendamment de son activité enzymatique, dans la régulation des premières étapes de différenciation des kératinocytes. En absence de LOX, l’initiation du programme de différenciation est perturbée, affectant la différenciation terminale et fonctionnelle des épidermes reconstruits. Ainsi, une régulation fine de l’expression de LOX est nécessaire au déroulement normal du processus de différenciation des kératinocytes, et donc au maintien de l’homéostasie épidermique / Lysyl oxidase (LOX) is an extracellular enzyme that catalyzes the cross-linking of fibrillar collagens or elastin, thereby regulating the structural integrity of connective tissues. Moreover, LOX displays multiple roles in different cell types, acting as a regulator of various biological processes at both extra and intracellular levels. The aim of the present work was to shed light on LOX functions in the epidermis, especially during keratinocyte differentiation. The development of culture models, consisting of confluent monolayers or reconstructed-epidermis allowed us to study LOX functions during the induction of the differentiation program, and furthermore during the terminal differentiation process leading to the formation of a pluristratified, cornified and functional epidermis. LOX expression is induced at the onset of the commitment to differentiation, both in primary and immortalized keratinocytes. Stable silencing of LOX expression affects the induction of the differentiation program and strongly impairs terminal and functional epidermal differentiation in reconstructed-epidermis. Therefore, LOX protein acts during the first steps of keratinocyte differentiation, independently of its enzymatic activity, and is implied for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is required for normal keratinocyte differentiation, and thus for maintenance of epidermal

Au sein d’une tumeur des régions hypoxiques se forment ce qui conduit à l’activation du facteur de transcription HIF1. HIF1, composé des deux protéines HIF1a et HIF1b, permet l’adaptation des cellules à des faibles concentrations d’oxygène en activant la transcription de gène cible. Un de ces gènes est celui codant pour la lysyl oxydase (LOX). Cette enzyme structure la matrice extracellulaire et est impliquée dans la tumorigénèse. Pour comprendre les liens entre LOX et HIF1a, nous avons modulé leurs expressions dans des lignées humaines de carcinome du côlon. En condition hypoxique, HIF1a contrôle l’expression de LOX et réciproquement, LOX régule la synthèse protéique d’HIF1a via l’activation de la voie de signalisation PI3K/AKT. Nous avons donc mis en évidence l’existence d’une boucle de régulation positive entre LOX et HIF1 en conditions hypoxique. Sachant que ces deux protéines sont des acteurs majeurs de la progression tumorale, nous avons cherché à comprendre le rôle de cette régulation mutuelle dans ce processus. Nos résultats démontrent que l’activité enzymatique de LOX promeut la croissance tumorale in vitro et in vivo et que son action est potentialisée par la présence de son partenaire HIF1a. De plus, LOX et HIF1a agissent en synergie afin d’augmenter la potentiel métastatiques des cellules tumorale de côlon in vitro. Ainsi, ce travail de thèse a permis de mettre en évidence l’existence d’une boucle de régulation entre HIF1a et LOX qui est critique dans la progression tumorale et semble également être impliquée dans le processus métastatiques / The microenvironment of solid tumors is exposed to hypoxic conditions which lead to the activation of Hypoxia‐Inducible Factor 1 (HIF1). HIF1, composed by a heterodimer of HIF1a and HIF1b protein, is a key transcription factor involved in cellular adaptation to changes in oxygen level, inducing the expression of several transcriptional targets such as Lysyl Oxidase (LOX). LOX is an amine oxidase that catalyzes crosslinking of fibrillar collagens and elastin in the extracellular matrix. Furthermore, LOX is implied in tumor progression. To clarify, the link between LOX and HIF1a, their expression were modulated in human colorectal carcinoma cell lines. We pointed out that besides HIF1‐dependant regulation of LOX, LOX can also act on the HIF1 pathway under hypoxic conditions. Indeed, LOX enzymatic activity upregulates HIF1a protein synthesis, and this action is mediated by the PI3K/AKT pathway. Thus, these results emphasize the existence of a regulation loop between HIF‐1a and LOX, which represent two main actors of tumoral progression. Thus, we wanted to determine the implication of this amplification loop in tumor progression. Our results show that LOX enzymatic activity increase tumor growth in vitro and in vivo, and this role is partially dependant of its partner HIF1a. Furthermore, we established that that LOX and HIF1a act in synergy to foster metastatic potential in colorectal carcinoma cell lines. Taken together, our results demonstrate a regulation loop between LOX and HIF1a with is critical for tumor progression and metastasis formation

El remodelat vascular es un procés crític que present en el desenvolupament de patologies cardiovasculars com l’aterosclerosi i l’aneurisma d’aorta abdominal (AAA). La Lisil Oxidasa (LOX) i la Fibulina-5 (FBLN5) són dues proteïnes de matriu extracel•lular essencials en la formació i manteniment de les fibres elàstiques. Ambdues participen en processos fisiopatològics caracteritzats per l’alteració de l’equilibri síntesi/destrucció de la matriu extracel•lular i han estat involucrades en el remodelat vascular. Estudis en models experimentals han proporcionat indicis sobre la possible vinculació d’aquestes proteïnes en el desenvolupament d’aterosclerosi i d’AAA, si bé existeixen molts aspectes de la biologia d’aquestes proteïnes que haurien de ser analitzats en profunditat. Un aspecte crític en el remodelat vascular associat a la progressió de la lesió ateroscleròtica és l’aparició de regions d’hipòxia. L’estrès hipòxic és característic d’alguns processos fisiològics, però també de patologies com el càncer, els trastorns isquèmics, la inflamació crònica i l’aterosclerosi. Les cèl•lules endotelials són els sensors primaris d’aquest estrés hipòxic, i la seva adaptació a la hipòxia es produeix a través d’una complexa resposta finament regulada que afecta múltiples aspectes de la seva biologia, com la supervivència cel•lular, el control del to i la permeabilitat vascular, l’angiogènesi i el remodelat de la matriu extracel•lular. No obstant, no s’ha establert si la hipòxia pot regular la expressió de la LOX i la FBLN5 en cèl•lules endotelials ni la repercussió fisiològica que podria tenir. Les investigacions desenvolupades pel nostre grup indiquen que la LOX és un enzim clau en el manteniment de la integritat de l’endoteli i de la funció endotelial. La capacitat d’aquest enzim de controlar l’expressió gènica, en base a la seva activitat al nucli, així com l’activitat de factors de creixement fonamentals en l’homeòstasi vascular com el bFGF i el PDGF, suggereix que aquest enzim controla funcions cel•lulars que poden ser determinants en el desenvolupament de malalties cardiovasculars. En base a aquests antecedents, ens hem plantejat com a hipòtesis de treball que la LOX i la FBLN5 són proteïnes clau en l’homeòstasi vascular, que participen en la resposta adaptativa de les cèl•lules vasculars a la hipòxia, i que contribueixen al desenvolupament de patologies vasculars com l’aterosclerosi i l’aneurisma d’aorta abdominal. Els nostres resultats han demostrat que en artèries coronàries humanes, la LOX s’expressa principalment a l’endoteli i a l’adventícia, i que és la isoforma més expressada en cèl•lules vasculars, juntament amb la LOXL2. En cèl•lules endotelials, l’expressió de LOX i FBLN5 s’incrementa en resposta a hipòxia a través de mecanismes transcripcionals, si bé amb clares diferències entre ambdues proteïnes. Així, el factor HIF-1 juga un paper secundari en la regulació de la LOX per hipòxia en cèl•lules endotelials, una resposta en la que estan involucrades proteïnes de la família Smad i l’estrès oxidatiu. Per contra, HIF-1 és el principal responsable de la inducció de la FBLN5 en cèl•lules endotelials, en les quals hem demostrat que aquesta proteïna contribueix a la resposta adaptativa a l’estrès hipòxic. Finalment, hem observat que la sobre-expressió de LOX en cèl•lules endotelials mitjançant un sistema lentiviral indueix canvis en l’expressió gènica que afecten la senyalització i la comunicació cèl•lula-cèl•lula, d’entre els quals en destaca la inhibició de l’α2-macroglobulina. / Vascular remodeling is a critical process in the development of cardiovascular diseases such as atherosclerosis and abdominal aorta aneurysm (AAA). Lysyl oxidase (LOX) and Fibulin-5 (FBLN5) are two extracellular matrix proteins essential for the elastic fiber development and maintenance, and they both have an active role in physiological processes in which vascular remodeling is involved. By using different animal models, these proteins have been suggested to be involved with atherosclerosis and AAA. A critical aspect of the vascular remodeling in the atherosclerotic lesion is the apparition of hypoxic areas. Endothelial cells are primary sensors to the hypoxic stress, responding to it in a complex but subtly regulated way that affects multiple aspects of its biology. Nevertheless, it has not been established if the hypoxic stimulus can modulate LOX and FBLN5 expression, nor its possible physiological repercussions. Research made in our group shows the importance of LOX in the endothelial activity and vascular function. LOX can control the genetic expression as well as the activity of growth factors essential for the vascular homeostasis, suggesting that this enzyme may control cellular functions essential for the development of cardiovascular diseases. Based on these results, we have considered as working hypothesis that LOX and FBLN5 are key proteins in the vascular homeostasis, that they take part of the vascular response to hypoxia, and that they contribute to the development of vascular diseases like atherosclerosis and the AAA. Our results show that hypoxia transcriptionally induces LOX and FBLN5 in endothelial cells. While HIF-1 plays a secondary role in the regulation of LOX, being also involved in this modulation Smad proteins and oxidative stress, it is the main factor responsible for the FBLN5 induction. We also demonstrate that the modulation of FBLN5 in endothelial cells contributes to their adaptive response to hypoxia. Moreover, we have observed that overexpressing LOX in endothelial cells downregulates α2-macroglobulin expression.

Abstract Erythropoiesis is the process of red blood cell production. The main regulator is the erythropoietin (EPO) hormone, which is strongly upregulated in low oxygen concentration (hypoxia) in cells via the hypoxia-inducible transcription factor HIF. The stability of HIF is regulated in an oxygen-dependent manner by three HIF prolyl 4-hydroxylases, all of which are known to participate in the regulation of erythropoiesis. A role in erythropoiesis of a fourth prolyl 4-hydroxylase, P4H-TM, which possesses a transmembrane domain, is not known, but it is able to hydroxylate HIF at least in vitro and in cellulo. The role of P4H-TM in erythropoiesis was studied by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null, Hif-p4h-3 null, and Hif-p4h-2 hypomorph mouse lines. The current study suggests that P4H-TM is involved in the regulation of EPO production, hepcidin expression and erythropoiesis. P4H-TM can thus be a new target for inhibition when designing novel pharmacological treatment strategies for anemia. LOX is required for crosslink formation between lysine residues in fibrillar collagens and elastin. These crosslinks enhance the tensile strength of collagen fibers and provide elasticity to elastic fibers and thus generate important structural support for tissues. LOX is required for normal embryonic development of the cardiovascular and pulmonary systems, and its depletion leads to a generalized elastinopathy and collagenolysis leading to perinatal death of Lox null mice. The development of muscles is a delicate process, which requires coordinated signaling and a homeostatic balance between the muscle and muscle connective tissue. Based on the drastic defects that were found in the present study in the skeletal muscle of Lox null mice, lack of LOX clearly disturbs this balance and increases transforming growth factor β (TGF-β) signaling, which leads to defects in the skeletal muscles. The impaired balance can cause muscle disorders, such as Duchenne Muscular Dystrophy (DMD). Despite the clinical significance, very little is known about the mechanisms controlling this homeostatic balance. The discovery of LOX as a regulating factor during skeletal muscle development will help to clarify the role of extracellular matrix (ECM) in muscle development and in muscle related congenital diseases. / Tiivistelmä Erytropoieesi on fysiologinen prosessi, jossa tuotetaan veren punasoluja ja jonka pääsäätelijänä toimii erytropoietiini (EPO) hormoni. EPO:n geeni ilmentyy voimakkaasti alhaisessa happipitoisuudessa (hypoksia) hypoksia-indusoituvan transkriptiotekijän (HIF) toimesta. HIF-tekijän stabiilisuutta säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4H) hapesta riippuvaisesti, ja niiden tiedetään siten osallistuvan myös erytropoieesin säätelyyn, HIF-P4H-2:n toimiessa pääsäätelijänä. Neljännen transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) roolia erytropoieesissa ei vielä tiedetä, mutta sen tiedetään säätelevän HIF-tekijää. Työssä käytettiin Hif-p4h-2, Hif-p4h-3 ja P4h-tm muuntogeenisiä hiirilinjoja, joiden entsymaattinen aktiivisuus on alentunut tai poistettu. P4H-TM:n osallisuutta erytropoieesin säätelyyn tutkittiin antamalla hiirilinjoille HIF-P4H-entsyymejä inhiboivaa lääkettä. Tutkimuksen tulokset osoittavat ensimmäistä kertaa P4H-TM:n säätelevän EPO-geenin ilmentymistä ja siten erytropoieesia. Ennestään tiedettyjen HIF-P4H entsyymien inhiboinnin lisäksi P4H-TM:n inhibointia voidaan pitää uutena kohteena uusien farmakologisten hoitokeinojen kehityksessä. Lysyylioksidaasi (LOX) katalysoi säikeisten kollageenien välisten sekä elastisten säikeiden välisten poikkisidosten muodostumista. Pokkisidokset antavat vetolujuutta kollageeneille ja joustavuutta elastisille säikeille ja ovat siten tärkeitä kudoksen rakenteelle. LOX:ia tarvitaan sikiön kehityksen aikana mm. hengitys-, sydän- ja verisuonielimistöjen kehityksessä. LOX:in puutos hiirillä aiheuttaa viallisia elastisia- ja kollageenisäikeitä, johtaen poikasten kuolemaan synnytyksen yhteydessä. Lihasten kehitys on tarkoin säädelty prosessi, jossa lihas ja lihaksen sidekudos säätelevät toisiansa. LOX:n suhteen poistogeenisissä Lox-/- sikiöissä löydettiin selviä ongelmia luurankolihasten kehityksessä. LOX:n puutoksen osoitettiin lisäävän transformoivan kasvutekijä beetan (TGF-β) määrää, joka estää luustolihaksia kehittymästä normaalisti. LOX kykenee sitoutumaan TGF-β:aan ja inhiboimaan sen aktiivisuutta ja LOX:n puuttuessa inhibointia ei tapahdu. Tutkimus osoittaa LOX:n olevan keskeinen tekijä lihaksen kehityksessä ja siten auttaa ymmärtämään sidekudoksen merkitystä luurankolihasten kehityksessä ja siihen liittyvissä sairauksissa.