Aortic valve stenosis (AS) development is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. Toll-like-receptor-3 (TLR3) is a lysosomal pattern-recognition receptor that binds double-stranded RNA and promotes pro-inflammatory cellular responses. In recent years, TLR3 has emerged as a major regulator of vascular inflammation. The exact role of TLR3 in the development of AS has not been investigated. Isolated human valvular interstitial cells (VICs) were stimulated with the TLR3-agonist polyIC and the resulting pro-inflammatory and pro-osteogenic response measured. Severe AS was induced in wildtype- and TLR3−/− mice via mechanical injury of the aortic valve with a coronary springwire. TLR3 activation was achieved by polyIC injection every 24 h after wire injury, while TLR3 inhibition was realized using Compound 4a (C4a) every 48 h after surgery. Endothelial mesenchymal transition (EndoMT) of human valvular endothelial cells (VECs) was assessed after polyIC stimulation. Stimulation of human VICs with polyIC promoted a strong inflammatory and pro-osteogenic reaction. Similarly, injection of polyIC marginally increased AS development in mice after wire injury. AS induction was significantly decreased in TLR3−/− mice, confirming the role of endogenous TLR3 ligands in AS pathology. Pharmacological inhibition of TLR3 with C4a not only prevented the upregulation of inflammatory cytokines and osteogenic markers in VICs, and EndoMT in VECs, but also significantly abolished the development of AS in vivo. Endogenous TLR3 activation significantly contributes to AS development in mice. Pharmacological inhibition of TLR3 with C4a prevented AS formation. Therefore, targeting TLR3 may be a viable treatment option.
ZusammenfassungDie kalzifizierende Aortenklappenstenose stellt die häufigste interventionell oder operativ behandlungsbedürftige Herzklappenerkrankung im Erwachsenenalter dar und betrifft insbesondere Menschen höheren Lebensalters. Obwohl der Fortschritt interventioneller Therapieoptionen die Behandlung der Aortenklappenstenose in den letzten Jahren verbessern konnte, ist die symptomatische Aortenklappenstenose weiterhin mit hoher Morbidität und Letalität assoziiert. Ihre Pathophysiologie ist gekennzeichnet durch eine Fibrosierung und Kalzifizierung der Klappentaschen, welche zu deren progredienter Verdickung und Versteifung und letztendlich zur Obstruktion der Klappe mit erschwertem Blutfluss in die Aorta führen. Da sich die Betroffenen meist im fortgeschrittenen Alter befinden und weitere Begleiterkrankungen aufweisen, ist ein operativer oder interventioneller Ersatz der Aortenklappe mit einem höheren Eingriffsrisiko und verlängerter Rekonvaleszenzzeit der Patienten verbunden. Die häufig begleitend auftretende periphere vaskuläre Verschlusskrankheit kann die Nutzung der peripheren Zugangswege im Rahmen des transluminalen Vorgehens unmöglich machen und die transapikale Punktion mit konsekutiv erhöhtem Eingriffsrisiko erfordern.Eine limitierte Lebenserwartung, z. B. im Rahmen von neoplastischen Erkrankungen, kann darüber hinaus eine Kontraindikation zur operativen und interventionellen Versorgung darstellen.Aktuell gibt es keine spezifische medikamentöse Therapie, die Initiation und Progression dieser bedeutenden Erkrankung beeinflussen kann und eine Alternative zum Klappenersatz für diese vulnerablen Patientenkollektive darstellt. Ein besseres Verständnis der zugrunde liegenden komplexen Pathophysiologie hat zur Entwicklung und Erprobung innovativer medikamentöser Therapieansätze geführt. Diese neuartigen Therapien befinden sich im Moment allesamt noch in Prüfung durch präklinische und klinische Studien und sollen in diesem Übersichtsartikel adressiert werden.
Background Calcific aortic valve stenosis (CAVD) is the most prevalent heart valve disease worldwide and, in advanced stages, leads to clinical deterioration and poor prognosis. Until now, no medical treatment is available. Thought to be a merely degenerative disease, we now know that disease initiation and progression are actively regulated by immune cell infiltration, chronic inflammation, osteogenic differentiation of valvular interstitial cells (VIC) and endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells (VEC). Suppressor of Cytokine Signaling 3 (SOCS3) is known to be a key regulator of inflammation. It promotes the polarisation of macrophages to an inflammatory phenotype and thus, contributes to disease progression in atherosclerosis. In CAVD, not only inflammation but also heterotopic bone formation leads to the thickening and obstruction of the aortic valve cusps and as SOCS3 is one of the main regulators of physiological bone formation, it could play a critical role in the progression of CAVD. Purpose We hypothesize that SOCS3 plays a crucial role during calcification of the aortic valve through regulation of EndMT, calcification of valvular interstitial cells and macrophage polarization. Methods and results In initial screening experiments, we investigated SOCS3-protein expression in explanted human aortic valves from patients undergoing surgical aortic valve replacement. SOCS3 is expressed in both stenotic and non-stenotic valve tissues. Immunofluorescence-staining of human aortic valves shows the co-localisation of SOCS3 with the interstitial cell-marker Vimentin and SOCS1, which is known to regulate macrophage-polarisation together with SOCS3 (Fig. 1). Staining of the macrophage-marker CD68 revealed its co-localisation with calcified areas of the aortic valve cusp. We validated our findings in our in vitro model of VIC calcification using two different calcifying conditions. Upregulation of RUNX2 and BMP2 verified successful osteogenic differentiation of VICs. During osteogenic differentiation for 7 days, SOCS3 and SOCS1 are significantly upregulated (Fig. 2A). We were able to induce EndMT by stimulating VEC with either TNFα or TGFβ/IL1β in vitro. We show that EndMT leads to the loss of endothelial cell markers like eNOS and VWF and to an upregulation of interstitial cell markers (such as Vimentin and α-SMA) and markers of EndMT (SNAI2). During EndMT, we were also able to observe a significant upregulation of SOCS1 and SOCS3 after 7 days (Fig. 2B). Ongoing knockdown experiments will help to elucidate the role of SOCS1 and SOCS3 during initiation and progression of CAVD. Conclusion We aim towards a better understanding of SOCS3 and its role in inflammation and calcification as hallmarks of the pathogenesis of CAVD. Since Zoledronic acid has been shown to induce a decreased expression of SOCS3 in macrophages, this might present as a possible treatment strategy to target SOCS3 therapeutically. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft
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