Unique features of GFAP expressing valve interstitial cells in valvular homeostasis and fibro-calcific aortic valve disease

Abstract Background The specialized three-layered microarchitecture of the aortic valve (AV) is designed to sustain extreme biomechanical forces throughout cardiac cycles. The extracellular matrix (ECM) of the AVs inner layer, the spongiosa, mediates and absorbs mechanical forces between layers and consists mostly of proteoglycans (PG) and glycosaminoglycans along with a valve interstitial cell (VIC) phenotype expressing glial fibrillary acidic protein (GFAP), an astrocyte marker. Purpose The role of GFAP+ VICs in maintaining functional and structural AV integrity and homeostasis and their pathological involvement in fibro-calcific AV disease (FCAVD) is characterized in the present study. Methods Human control and FCAVD tissues and mammalian AVs were analyzed using pentachrome histological and immunohistochemical staining and western blotting for GFAP protein abundance. Whole mount immunostaining and optical clearing was used to evaluate the 3D intra-leaflet GFAP distribution in non-calcified (n=3) and FCAVD patients (n=3). Spatial transcriptomics of GFAP+ areas in non-calcified AVs (n=5) from male patients with either aortic dissection (AD) or AV regurgitation (AI) and male FCAVD patients (n=5) was performed. Results GFAP was expressed in all human heart valves and in mammalian AVs. Semilunar valves, AV and pulmonary valve, showed a 10-fold higher GFAP protein abundance than the atrioventricular valves (mitral and tricuspid valves) p≤0.04). 3D visualization of GFAP distribution revealed a homogeneous GFAP distribution across the spongiosa of non-calcified AVs, which was pathologically remodeled in in FCAVD. While in non-calcified AVs GFAP+ and GFAP- areas showed highly divergent gene expression patterns, this diversity was reduced 4-fold in AVs from FCAVD patients. Only 17 genes in GFAP+ areas and 5 genes in GFAP- areas remained overrepresented, indicating active modulation of complement system components towards opsonization and apoptotic cell clearance, ECM remodeling, lipid metabolism, angiogenesis, cell death, inflammatory responses and cell survival. Conclusion The present study shows that GFAP+ VICs are evolutionarily conserved across mammalian species. GFAP+ VIC gene expression patterns differ significantly from GFAP- VICs, suggesting that GFAP+ VICs provide essential functions for tissue homeostasis, which is strongly affected in FCAVD. As PLA2G2A is the most robust candidate consistently associated to GFAP gene expression and significantly enriched in FCAVD, involvement of GFAP+ VICs in modulation of inflammatory responses and lipid remodeling is conceivable.