Crosstalk between myofibroblasts and macrophages: A regulative factor of valvular fibrosis in calcific aortic valve disease

Inflammation and fibrosis are highly correlated with the progression of calcific aortic valve disease (CAVD). As one of the differentiated forms of valvular interstitial cells, myofibroblasts play a critical role in CAVD's development as do macrophages. Although numerous studies have been conducted on them separately, their communication and interaction remain unclear. We used porcine aortic valves to isolate valve interstitial cells (VICs). VICs were induced to differentiate into myofibroblasts by transforming growth factor-beta 1 (TGF-beta 1). After successful activation was determined, the myofibroblast-conditioned medium (CM) was collected and used to act on RAW264.7, a macrophage cell line. A migration and adhesion assay estimated the recruitment capability of myofibroblasts on macrophages. We used flow cytometry, quantitative polymerase chain reaction (qPCR), and Western blot analysis to investigate myofibroblasts' polarity promotion function in macrophages. Finally, we used macrophage-CM on VICs to explore the differentiation induction function of polarized macrophages. Myofibroblast marker alpha-smooth muscle actin and M2 macrophage marker CD163 were detected as upregulated in CAVD patients, and their expression has a certain correlation. The Smad3/HA/CD44 axis activated the differentiation of myofibroblasts by Western blot. The myofibroblast-CM can promote chemotaxis and adhesion of macrophages through protein kinase B/chemokine (C-C motif) ligand5 and Smad3/HA/CD44, respectively. Hyaluronic acid (HA) inside the myofibroblast-CM stimulates macrophages to polarize into M2 macrophages. In turn, M2 macrophage-CM has the promotive ability to activate myofibroblasts but fails to induce the osteoblast differentiation of VICs directly. The crosstalk between myofibroblasts and macrophages causes the excessive activation of myofibroblasts. This positive feedback loop may play a vital role in CAVD progression.