Macrophages Promote Aortic Valve Cell Calcification and Alter STAT3 Splicing

Abstract

Objective: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1 +/− model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1 +/− murine aortic valves were characterized by flow cytometry. Macrophages in Notch1 +/− aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1 +/− macrophages drive disease. Notch1 +/− mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1 +/− aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3β isoform. Conclusions: This study reveals that Notch1 +/− aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

Interferons Are Pro-Inflammatory Cytokines in Sheared-Stressed Human Aortic Valve Endothelial Cells

“…In this study, initial experiments under static conditions revealed that human VEC sense type I and II IFNs, which triggers the activation of STAT1 and NF-κB, and the subsequent expression of inflammatory mediators. Additional pathways, like STAT3, could also play a role in JAK/STAT signaling in valve cells, given that macrophage recruitment promotes calcification and alters STAT3 splicing in the Notch1(+/−) model of CAVD [ 34 ]. The disease occurs in a side-specific manner, likely mediated by the significant differences in hemodynamic forces experienced by each side of the aortic valve [ 1 , 4 , 22 ].…”

“…Our data suggest a potential interplay between inflammatory cytokines and oscillatory flow on monocyte adhesion that may be relevant to CAVD. In fact, infiltrated monocyte-derived cells, such as macrophages, are enriched in calcified valves and show increased inflammatory and osteogenic activity [ 34 , 49 , 50 ]. Moreover, the enrichment of polarized macrophages in bicuspid valves and the activation of macrophages by cyclic mechanical strain in several models suggest a potential role for mechanical strain in macrophage activation and the ensuing inflammation in CAVD [ 49 ].…”

Genetic ablation of serotonin receptor 2B improves aortic valve hemodynamics of Notch1 heterozygous mice in a high-cholesterol diet model

“…Our data show Htr2b ablation nor receptor inhibition alter Runx2 expression or osteogenic calcification, suggesting the hemodynamic phenotype observed in mutant mice is not based on canonical osteogenic signaling pathways (Fig 6). Immune cell recruitment plays a key role in other 5-HT 2B -mediated fibrotic disease and has been shown to contribute to the Notch1 +/model of CAVD, suggesting it may be involved here [25,30,39]. This may also indicate a phenotype arising from cells other than myofibroblasts.…”

“…These clinical and basic research findings led us to hypothesize that 5-HT 2B may be a novel target for pharmacological treatment of CAVD. Therefore, we evaluated the impact of both genetic and pharmacological ablation of 5-HT 2B in the well-established Notch1 +/mouse model of CAVD, based on the heritable form in patients with NOTCH1 mutations [21][22][23][24][25]. Briefly, we found that genetic ablation mitigated CAVD phenotypes, while pharmacological targeting was not efficacious.…”

New calcification model for intact murine aortic valves

“…Aside from the phosphates required to generate mineral depositions, inorganic phosphates have been shown to regulate expression of proteins such as BMP2 and osteopontin [31,32]. Studies using in vitro models have demonstrated the important role of mechanical stress, the influence of the matrix and the interaction between VICs, VECs and macrophages in the process of calcification [5,7,[33][34][35][36][37]. These studies thereby demonstrated that the absence of the complete valvular structure might prevent drawing accurate conclusions for in vivo calcification.…”