Hypoxia Promotes Human Umbilical Vein Smooth Muscle Cell Phenotypic Switching via the ERK 1/2/c-fos/NF-κB Signaling Pathway

Abstract

Vein wall hypoxia has long been suggested as a key factor for the development of varicose veins (VVs) and accumulating evidence has revealed the phenotypic transformation of vascular smooth muscle cells (VSMCs) under hypoxic conditions. However, the underlying molecular mechanisms of this process remain poorly understood. Our previous study revealed a positive correlation between c-fos expression and VSMC functional disturbance of VVs. This study aimed to further explore the role of c-fos in the phenotypic switching of VSMCs under hypoxic conditions. Human umbilical vein smooth muscle cells (HUVSMCs) were cultured under hypoxia or normoxia. PD0325901 (10 μmol/L) and pyrrolidine dithiocarbamate (PDTC) (10 μmol/L) were used to inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κ B (NF-κB) signaling pathways, respectively. HUVSMCs stably overexpressing c-fos were constructed to explore the underlying mechanism. The Western blot analysis was performed to detect the protein expression levels of c-fos, phosphorylated p65 (p-p65), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), osteopontin (OPN), and α-smooth muscle actin (α-SMA). Cell proliferation and migration capacity were detected by a Cell Counting Kit 8 (CCK-8) assay and a wound-healing assay, respectively. The cell apoptotic rate was determined using the Annexin V-FITC Apoptosis Detection Kit. Hypoxic exposure increased the expression levels of indicators of the p-ERK1/2/c-fos and NF-κB signaling pathways, which was accompanied by altered levels of phenotypic biomarkers (α-SMA and OPN). Cells exposed to hypoxia were characterized by a greater proliferative and migratory ability. No significant differences were observed in the rate of cell apoptosis between the normal group and the hypoxic group. In addition, inhibition of the ERK1/2/c-fos signaling pathway by PD0325901 (10 μmol/L) reduced the expression of inflammatory cytokines and attenuated hypoxia-mediated phenotypic transformation. Furthermore, inhibition of the NF-κB signaling pathway by PDTC (10 μmol/L) downregulated the expression level of OPN and reduced the migration of HUVSMCs under hypoxia exposure. However, pretreatment with PDTC did not suppress the expression of c-fos or cell proliferation. Finally, the introduction of exogenous c-fos in HUVSMCs induced increased protein expression levels of p-p65, COX-2, and OPN, accompanied by a remarkable increase in HUVSMC proliferation and migration. Our research demonstrated that hypoxia could promote the phenotypic transformation of HUVSMCs partially through the ERK1/2/c-fos/NF-κB signaling pathway, which provided a novel insight into hypoxia-associated venous wall remodeling to furtheraid the development of a novel therapeutic target for the prevention or treatment of VVs.