Contribution of hypoxia inducible factor, HIF1α, to vascular inflammation and remodeling in giant cell arteritis (GCA). Effects of GM-CSF receptor blockade on HIF1α stabilization

Abstract

Giant Cell Arteritis (GCA) is an immune-mediated disease affecting large and medium sized arteries. Its pathophysiology is not fully understood and its present treatment relies on glucocorticoids, which have important side effects. Therefore, studying the disease and finding new treatments is an unmet medical need. GCA lesions undergo several changes during the development of the disease. Thickening and remodeling of the vascular wall are due to the aberrant inflammatory infiltrate. These changes promote angiogenesis. Angiogenesis is one of the main consequences of hypoxia, through stabilization and induction of Hypoxia Inducible Factor 1 alpha (HIF1α). Several inflammatory molecules such as IL6, IL1β, TNFα and IFNγ, present in GCA lesions, have been demonstrated to be able to stabilize HIF1α. Moreover, several HIF1α target genes like IL6 and other proinflammatory molecules are present in GCA lesions. Moreover, vascular smooth muscle cells (VSMC) are able to express HIF1α under certain conditions. GM-CSF is an important inflammatory mediator expressed by several inflammatory and vascular resident cells. It downstream pathway includes JAK/STAT activation. Regarding GCA, GM-CSF can be affecting several important points of its pathogenesis, from initial dendritic cells activation to leucocyte recruitment and giant cell formation. Considering all these together, our hypothesis for the present thesis is as follows: HIF1⍺ and GM-CSF play an important role in vascular inflammation and remodeling in GCA, through independent or inter-related mechanisms. The objectives are to determine the presence/absence of HIF1α in GCA lesions as well as triggers and consequences of it. To analyze the effect of anti-cytokine targeted therapies in this pathway. To investigate GM-CSF pathway in GCA and the effect of its blockade with mavrilimumab. In the present thesis we demonstrate the presence of hypoxia in the GCA lesions as well as the presence of HIF1α in the media layer of GCA temporal arteries. We validate that VSMC, the cells present in the media layer of the artery, are able to stabilize HIF1α under an inflammatory microenvironment with an in-vitro model of the disease. Under this inflammatory microenvironment VSMC also increase the expression of GLUT1 and PHD3 transcripts, which are well-described HIF1α target genes indicating HIF1α functional activity in these cells. VSMC modify its transcriptome when HIF1α is stabilized affecting important pathways in GCA pathogenesis. In these cells the migratory capacity is reduced, angiogenic factors are increased (VEGFA, IL6, CXCL8), chemokine expression is imbalanced (CCL2 and CCL5 decreased and CXCL8 increased) and inflammation-relation transcripts were increased (IL6, IL1B, TNFA and CSF2). Inhibiting TNFα, IL1 and GM-CSF in an inflammatory microenvironment was able to reduce HIF1α protein levels in VSMC. GM-CSF and its receptor (GM-CSFRα) are present in GCA lesions, and its downstream pathway (JAK2/STAT5) is activated. Inhibiting GM-CSF effects with mavrilimumab, a monoclonal antibody against GM-CSF receptor alpha chain , in an ex-vivo model of the disease induced the following effects: reduction of lymphocyte and myeloid cell markers; reduction in T cell activation related molecules and Th1 differentiation pathway; decrease of pro-inflammatory cytokines; decrease of mediators of vascular injury; and reduction in tissue angiogenesis. Blocking GM-CSF receptor with mavrilimumab reduces some HIF1α targets (IL6, IL1β, and TNFα) which, including IFNγ, are also identified as stimuli for HIF1α that accumulated in VSMC, supporting the relationship between HIF1α and GM-CSF pathways in the pathogenesis of GCA.

La fisiopatologia de l’arteritis de cèl·lules gegants (ACG) no es coneix amb profunditat i el tractament actual està basat en glucocorticoides amb els seus afectes adversos pel que és necessari seguir estudiant la malaltia i trobar millors teràpies. Les artèries dels pacients amb ACG experimenten un engruiximent i remodelat de la paret degut a un infiltrat inflamatori aberrant. Aquests canvis promouen angiogènesi, una de les principals conseqüències de la hipòxia, mitjançant l’HIF1α. Hi ha diverses molècules inflamatòries presents en les lesions descrites com estabilitzadores de l’HIF1α. A més a més, diverses molècules diana d’aquest factor es troben presents en les lesions. Uns del tipus cel·lulars importants en la fisiopatologia, les cèl·lules muscular llises (VSMC) poden expressar l’HIF1α i durant el desenvolupament de l’ACG adquireixen un fenotip pro-inflamatori. Tot això, fa que una hipòtesi raonable sigui que l’HIF1α té un rol important en la fisiopatologia de l’ACG i en el desenvolupament i manteniment de la inflamació en les lesions. En aquesta tesi es demostra hipòxia i la presencia d’HIF1α en les lesions d’ACG. L’HIF1α es troba principalment en la capa mitja de la paret vascular on es troben les VSMC. També es demostra que les VSMC són capaces d’estabilitzar l’HIF1α quan es troben en un microambient inflamatori; i en aquestes condicions també augmenta l’expressió dels gens diana de HIF1α. Quan estabilitzen in-vitro l’HIF1α en les VSMC aquestes canvien la seva expressió de diversos gens i proteïnes implicats en el remodelat vascular, l’angiogènesi i la quimiotaxi. Sota el microambient inflamatori, quan certes molècules pro-inflamatòries (GM-CSF, IL1β i TNFα) es troben inhibides els nivells d’HIF1α disminueixen. En concret, la inhibició del receptor del GM-CSF mitjançant l’ús del mavrilimumab, disminueix diverses molècules inflamatòries, angiogèniques i de dany vascular. Algunes d’aquestes molècules estan descrites com a inductores i alhora gens diana de l’HIF1α. En resum, es demostra que l’HIF1α juga un paper important en la fisiopatologia de l’ACG, així com el GM-CSF i que ambdós factors es troben estretament relacionats en aquesta malaltia.