Endothelial and myointimal cell functions related to vascular inflammation and remodeling in Giant-Cell Arteritis: Contribution of interleukin-23p19, interleukin-35 and endothelin-1

Abstract

Giant cell arteritis (GCA) is vascular inflammatory disease frequently involving large and medium sized arteries. GCA pathogenesis is not completely characterized, although there is a well accepted general model based on the observation of the histopathology and supported by immunopathology studies. According to this model, CD4+T cells subsets (mainly Th1 and Th17) and macrophages are essential to orchestrate the amplification of pro-inflammatory cascades leading to chronic inflammation of the vessel wall. The main sites of entry of infiltrating leukocytes are the activated endothelial cells (EC) from the adventitial vasa vasorum. Importantly, the secretion of proteases by macrophages among others, promotes elastic lamina degradation and migration of leukocytes to the inner artery layers. Vascular smooth muscle cells (VSMC) have also an important role in GCA pathogenesis since, in this scenario; they acquire a pro-inflammatory phenotype by overexpressing adhesion molecules and pro-inflammatory cytokines, contributing to the persistence of inflammation. Inflammatory cell products, as well as vascular injury, trigger a vascular remodelling process which leads to the development of intimal hyperplasia and vascular lumen occlusion. VSMC acquisition of proliferative and migratory phenotype may contribute to vascular remodeling. Further investigation of molecules involved in GCA pathogenesis is needed in order to find new strategies to improve the current GCA treatment (glucocorticoids) since 50% of the patients relapse and 40% suffer form early (visual loss or stroke) or late (aortic aneurysm) complications derived from maladaptive vascular remodeling. For this reason, the aim of this doctoral thesis was to contribute knowledge to these GCA challenges by investigating new roles of two important molecules involved in the persistence of inflammation (IL-23p19 and IL-35) as well as a new role of endothelin 1 (ET-1) in GCA vascular remodeling.

The IL-12 superfamily of cytokines is composed by four heterodimeric cytokines: IL-12 (p40 + p19), IL-23 (p40 ad p19), IL-27 (p28 and Ebi3) and IL-35 (Ebi3 and p35). Regarding to these cytokines, we have demonstrated a dichotomy in the expression of p19 and p40 subunits suggesting a role for p19 independent from p40 heterodimerization. We observed that p19 but not p40 was induced by a pro-

inflammatory context in EC from the adventitial vasa vasorum of GCA specimens. Moreover, p19 was not secreted by stable p19-expressing EC suggesting an intracellular role. Our studies showed that p19 contributed to leukocyte attachment and transmigration by promoting overexpression of adhesion molecules ICAM-1 and VCAM-1. We also demonstrated that these effects were triggered by intracellular p19 binding to gp130 receptor, subsequently promoting phoshporylation and nuclear translocation of STAT3.

IL-35 cytokine was also investigated as a consequence of the previous observation that p35 (but not p40) was constitutively expressed by VSMC of GCA specimens. This dichotomy suggested that p35 may have and independent role from that derived from p40 heterodimerization or that p35 may have another partner to form a cytokine different from IL-12. Our results demonstrated that p35 dimerizes with Ebi3 subunit in order to form IL-35 in GCA specimens. Interestingly, we found that IL-35 was mainly produced by leukocytes and VSMC in the adventitial/media junctions of GCA lesions. In order to mimic this context, we used co-cultures of temporal artery-derived primary VSMC together with leukocytes which demonstrated that IL-35 expression in VSMC was increased by pro-inflammatory cytokines IL-1β, TNFα and IFNg. In addition, we have demonstrated a new role for this cytokine in VSMC. IL-35 interacts with gp130 in order to promote pro-inflammatory phenotype of VSMC. This is an important observation since, until present; IL-35 has been described as an anti-inflammatory cytokine involved in regulatory T cell (Treg) differentiation.

We previously reported that endothelin-1 (ET-1) and its receptors ETAR and ETBR are over-expressed in GCA lesions and GCA patients with ischemic complications have increased serum concentrations of ET-1. Although it has been observed that ET-1 may contribute to myofibroblast differentiation of fibroblasts, a crucial step in lung and skin fibrogenic diseases, the most investigated function of ET-1 in VSMC has been vascular tone regulation. In this thesis, we have demonstrated that ET-1 may contribute to vascular occlusion, by promoting the migration of VSMC through activation of the focal adhesion kinase (FAK)/Src and PI3K axis. In GCA lesions ET-1 was mainly produced by inflammatory cells and the interaction with PBMC increased ET-1 receptor expression by VSMC. ET-1 stimulated VSMC migration by inducing focal adhesion kinase (FAK) phosphorylation at Y397 permitting association with p85 subunit of PI3Kinase which co-localized with FAK at the cell protrusions of

migrating cells. Accordingly, inhibition of both FAK and PI3K abrogated ET-1 induced migration. Consistently, blockade of ETAR and ETBR with BQ123 and BQ788 antagonists inhibited ET-1 induced VSMC migration and VSMC outgrowth from cultured GCA-arteries.

L’arteritis de cèl·lules gegants (ACG) és una malaltia inflamatòria crònica d’etiologia desconeguda que majoritàriament afecta les arteries de mitjà i gran calibre, entre elles l’artèria temporal. El tractament actual de l’ACG són els glucocorticoides tot i que el 50 % dels pacients recauen.

Els mecanismes implicats en l’ACG s’han anat caracteritzant a partir d’observacions histopatològiques de l’artèria temporal així com a partir d’estudis immunopatològics. S’observa un clar infiltrat inflamatori format majoritàriament per limfòcits CD4+T i macròfags que van migrant des de l’adventícia fins a la íntima. La porta d’entrada d’aquest infiltrat es creu que podrien ser els petits vasos adventicials (vasa vasorum) que en condicions normals nodreixen l’artèria i, en canvi, en condicions patològiques s’observa una clara activació de les cèl·lules endotelials que els composen. Les cèl·lules infiltrants secreten citoquines i quimiocines encarregades de reclutar constantment més leucòcits donant lloc a la persistència de la inflamació en el teixit arterial. A més, aquestes cèl·lules també interaccionen amb altres components de la paret arterial com les cèl·lules muscular llises (VSMC) que també adquireixen un fenotip pro-inflamatori.

Per altra banda, en l’ACG també s’observa un important remodelat vascular patològic, majoritàriament degut al trencament de les làmines elàstiques de l’artèria i la migració cap a la llum vascular tant de l’infiltrat inflamatori com de les VSMC que adquireixen un fenotip més proliferatiu, migratori i productor de matriu extracel·lular que ajudarà a crear la hiperplàsia de la íntima, originant l’oclusió vascular.

Tenint en compte aquest context, en aquesta tesi doctoral s’han estudiat dues molècules implicades en la persistència de la inflamació. Per una banda, l’estudi de la subunitat p19 de la IL-23. En condicions pro-inflamatòries aquesta molècula podria actuar independentment de la dimerització amb la p40 per formar la IL-23 i podria intervenir desde el compartiment intracel.lular a traves d’una interacció directa amb gp130, en l’activació de les cèl·lules endotelials que formen el vasa vasorum adventicial, ajudant així al reclutament leucocitari. Per altra banda, l’estudi de la IL-35, expressada per les VSMC i els leucòcits infiltrants i no només per les cèl·lules T reguladores com s’havia descrit anteriorment. Hem observat i descrit per primera vegada, que la IL-35 també estaria involucrada en la persistència de la inflamació de l’ACG, induint un fenotip pro-inflamatori en les VSMC.

Pel que fa al remodelat vascular, hem estudiat l’endotelina (ET-1), que a través dels seus dos receptors (ETAR i ETBR) provocaria una inducció de la migració de les VSMC, tenint un rol essencial en la creació de la hiperplàsia de la íntima i la oclusió vascular.