ML-7

Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes.

The Pm–human oral keratinocyte (HOK) co‐culture model was established to detect monolayer permeability, zona occludens‐1 (ZO‐1) expression, and intracellular survival of Pm. We performed RNA‐seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML‐7 was utilized in Pm‐HOK co‐culture model to assess its effects on monolayer permeability and ZO‐1 expression.

HOK monolayer permeability was increased, and ZO‐1 expression was decreased after co‐culture (p < 0.05). Pm could survive in HOK cells. RNA‐seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm‐HOK co‐culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO‐1 expression was upregulated (p < 0.05).

MLCK may be involved in disrupting epithelial barrier function by Pm.

Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis.

We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)^ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs.

The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.