Sutural fibroblasts exhibit the function of vascular endothelial cells upon mechanical strain

Abstract

Midfacial hypoplasia is a type of facial dysplasia. The technique of trans-sutural distraction osteogenesis promotes midface growth so as to ameliorate this symptom. In the process of distraction osteogenesis, the fiber matrix in the suture acts as a mechanical sensor. Compared with osteogenesis, the formation of collagen fibers by fibroblasts is significant in the early stage of sutural distraction. However the transformation of fibroblasts during sutural bone formation induced by tensile force is poorly characterized. Here, we used single-cell RNA sequencing to define the cell classification of the zygomatic maxillary suture and the changes of cell clusters in the suture before and after seven-day distraction. We identified twenty-nine cell subsets spanning monocyte/macrophages, neutrophils, red blood cells, B cells and fibroblasts. Compared with the control group, Monocle analysis revealed the emergence of a unique fibroblast subset (Cdh5+, Col4a1+, Fat1-, and Acta2-) (cluster 27) that expressed vascular endothelial cell genes within the distracted zygomatic maxillary suture. We constructed the differentiation trajectories of the fibroblast population (cluster 23, 27) in the suture before and after distraction. In addition, we clarified that a subset of fibroblasts (cluster 27) lost expression of Fat1, an upregulator of the Hippo pathway, and upregulated Cyr61, a downstream gene of the Hippo pathway, during the distraction process. Further enrichment analysis suggests that cells of the new subset (cluster 27) are undergoing conversion of their identity into a vascular endothelial cell-like state in response to mechanical stimulation, associated with upregulation of angiogenesis genes along the single-cell trajectory. Further immunofluorescence staining confirmed this phenomenon. A combined general transcriptome RNA sequencing data analysis demonstrated that the fibroblasts expressed a number of extracellular matrix-related genes under mechanical strain. These data together provide a new view of the role of fibroblasts in tension-induced sutural angiogenesis via interaction with the Hippo pathway.

Keywords: Angiogenesis; Fibroblasts; Hippo; Mechanic; Suture.