SVEC4-10

3-BrFlu Induces both MAPK Phosphorylation and Intracellular ROS Accumulation in SVEC4-10 Endothelial Cells

3-Bromofluoranthene (3-BrFlu), a PAH derivative, is implicated in endothelial damage through pro-inflammatory pathways. Li's team utilizes SVEC4-10 cell lines and zebrafish models to investigate 3-BrFlu's impact on vascular endothelial dysfunction, specifically examining the role of the MAPK-mediated NF-κB pro-inflammatory pathway and intracellular ROS generation.

MAPK phosphorylation of p38 MAPK, ERK and JNK functions as a primary upstream regulator for NFκB p65. Immunoblotting analysis revealed that 3-BrFlu triggers MAPK phosphorylation starting at 3 μM concentration and this effect becomes significant at that level (Fig. 1). Phosphorylation levels of p38 MAPK rose from 1.86 to 2.69 times, ERK levels increased from 1.31 to 1.94 times, and JNK levels elevated from 1.77 to 2.73 times when treated with 3–100 μM 3-BrFlu. Intracellular ROS which are essential for maintaining vascular barrier functionality demonstrated concentration-dependent accumulation beginning at 3 μM as detected by a DCFH-DA fluorescent probe (Fig. 2). The fluorescence intensity of ROS demonstrated an increase from 1.04 × 10⁴ to 1.83 × 10⁴–3.56 × 10⁴ when exposed to concentrations ranging from 10 to 100 μM of 3-BrFlu. The experimental results show that 3-BrFlu causes both MAPK phosphorylation and intracellular ROS accumulation in SVEC4-10 endothelial cells.

Fig. 1. 3-BrFlu-induced MAPK phosphorylation in SVEC4-10 endothelial cells (Lee C Y, Wu SW, et al., 2024).

Fig. 2. 3-BrFlu-induced intracellular ROS accumulation in SVEC4-10 endothelial cells (Lee C Y, Wu SW, et al., 2024).

LPA Receptor Expressions and Cell Growth Activity of SVEC4-10 Cells

LPA receptor signaling is crucial in cancer development, influencing cell migration and metastasis within the complex tumor microenvironment, which includes lymphatic endothelial cells. Hypoxia, common in solid tumors, further enhances tumor progression. Yamamoto et al. examined the effects of LPA receptor-mediated signaling on SVEC4-10 lymphatic endothelial cells under hypoxic conditions via in vitro assays. They evaluated changes in cell motility, tube formation, and responses to LPA agonists and antagonists.'

Six G protein-coupled LPA receptors exist, with LPA1, LPA2, and LPA3 belonging to the Edg family, while LPA4 to LPA6 are non-Edg. This study focused on LPA1, LPA2, and LPA3 to explore their roles in regulating hypoxia-induced functions of SVEC4-10 cells. SVEC4-10 cells expressed all LPA receptors except Lpar5 (Fig. 3A). Their growth rate increased under 1% O2 compared to 21% O2 (Fig. 3B), but Lpar1, Lpar2, and Lpar3 expression was lower at 1% O2 (Fig. 3C). LPA did not affect growth under 21% O2 but inhibited it at 1% O2 with 1 μM LPA (Fig. 3D). LPA2 agonist GRI-977143 inhibited growth, while LPA3 agonist (2S)-OMPT enhanced it (Fig. 3E). LPA1 antagonist AM966 increased growth with LPA (1 μM) (Fig. 3F). Thus, LPA1 and LPA2 suppress growth, whereas LPA3 promotes it under hypoxia.

Fig. 3. LPA receptor expressions and cell growth activity of SVEC4-10 cells (Yamamoto M, Takai M, et al., 2024).