Secreted Factors From Proinflammatory Macrophages Promote an Osteoblast-Like Phenotype in Valvular Interstitial Cells

Abstract

Objective: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified that 2 cytokines in M1 media attributed to the observed antifibrotic effects were TNF (tumor necrosis factor)-α) and IL (interleukin) 1β). After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. Conclusions: Proinflammatory cytokines in M1 conditioned media, notably TNF-α, IL-1β, and IL-6, inhibit the myofibroblast response in VICs and promote their osteogenic differentiation. Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.

miRNA Expression Profiling Uncovers a Role of miR-139-5p in Regulating the Calcification of Human Aortic Valve Interstitial Cells

“…The expression level of IL-6 was increased in calcified human aortic valves (Li et al, 2017b). Recombinant IL-6 can increase the expression of RUNX2 and osteopontin to promote the osteogenic differentiation of VICs; when IL-6 was inhibited by siRNA, the osteogenic differentiation of VICs was blocked in vitro (El Husseini et al, 2014;Grim et al, 2020). However, there is still no evidence for the function of miR-149-5p in osteogenic differentiation of VICs and CAVD and it could be explored in a future study.…”

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

“…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 ].…”

Network Modeling Predicts Personalized Gene Expression and Drug Responses in Valve Myofibroblasts Cultured with Patient Sera

“…Preventing valve fibrosis during AVS is crucial for long-term patient outcomes even after valve replacement interventions. Although previous studies have shown that alterations in serum protein levels after valve replacement can deactivate myofibroblasts 12,13 , the underlying mechanisms of this deactivation remain unclear due in part to the complexities of intracellular regulatory mechanisms. Using coupled serum proteomic and myofibroblast transcriptomic datasets for patients undergoing TAVR, we developed a strategy to integrate this patient-specific data into a computational model of myofibroblast signaling and transcriptional regulation to predict fibrosis-related protein expression in response to environmental signals.…”

“…In a similar finding, Hilfiker-Kleiner and colleagues found decreases in ERK and Akt phosphorylation for mice carrying a point mutation in gp130 and subjected to MI compared to wild-type mice. Recent evidence of IL6 as a promoter of VIC osteogenic differentiation via the increased expression of RUNX2 and osteopontin suggest that this pathway may serve multiple phenotypic roles 13 , and future targeted investigations of this behavior may explain possible context-dependent roles of IL6 signaling during AVS progression.…”

“…These extracellular cues are transduced through a complex network of regulation in which multiple intracellular signaling pathways interact via crosstalk and feedback mechanisms to dictate overall cell responses 11 , thus further complicating efforts to understand how myofibroblasts respond within the valve microenvironment. While previous studies have shown that myofibroblasts can undergo deactivation in response to serum biomarkers after TAVR, macrophage-secreted inflammatory cues, and local mechanical stiffness [12][13][14][15][16] , the complexities of myofibroblast signal transduction among a diverse microenvironment complicates efforts to control cell deactivation.…”