Background. Oscillatory shear stress (OSS) disrupts endothelial homeostasis and promotes oxidative stress, which can lead to atherosclerosis. In atherosclerotic lesions, Toll-like receptor 4 (TLR4) is highly expressed. However, the molecular mechanism by which TLR4 modulates oxidative changes and the cell signaling transudation upon OSS is yet to be determined. Methods and Results. Carotid artery constriction (CAC) surgery and a parallel-plate flow chamber were used to modulate shear stress. The results showed that OSS significantly increased the oxidative burden, and this was partly due to TLR4 activation. OSS activated NOX2 and had no significant influence to NOX1 or NOX4 in endothelial cells (ECs). OSS phosphorylated caveolin-1, promoted its binding with endothelial nitric oxide synthase (eNOS), and resulted in deactivation of eNOS. TLR4 inhibition restored levels of nitric oxide (NO) and superoxide dismutase (SOD) in OSS-exposed cells. Conclusion. TLR4 modulates OSS-induced oxidative stress by activating NOX2 and suppressing eNOS.

Cardiovascular disease is the highest cause of death, and atherosclerosis (AS) is the primary pathogenesis of many cardiovascular diseases. In this study, we aim to investigate the possible pharmaceutical effects of Dendrobium huoshanense C. Z. Tang et S. J. Cheng polysaccharide (DHP) in AS. We fed zebrafish with high-cholesterol diet (HCD) to establish a zebrafish AS model and treated with DHP and observed plaque formation and neutrophil counts under a fluorescence microscope. Next, a parallel flow chamber was utilized to establish low shear stress- (LSS-) induced endothelial cell (EC) dysfunction model. We observed that DHP significantly improved HCD-induced lipid deposition, oxidative stress, and inflammatory response, mainly showing that DHP significantly increased superoxide dismutase (SOD) activity, decreased plaque formation, and decreased neutrophil recruitment and the levels of total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), and reactive oxygen species (ROS). Furthermore, DHP significantly improved LSS-induced oxidative stress and EC dysfunction. Our results indicated that DHP can exert treatment effects on AS, which may attribute to its hypolipidemic, antioxidant, anti-inflammatory activities and improving LSS-induced EC dysfunction. DHP has promising potential for further development as a functional natural medicine source targeted at AS prevention.

BACKGROUND: In-stent restenosis is characterized by a significant reduction in lumen diameter within the stented segment, primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMCs) and neointimal hyperplasia. PFN1 (profilin-1), an actin-sequestering protein extensively studied in amyotrophic lateral sclerosis, remains less explored in neointimal hyperplasia. METHODS: Utilizing single-cell RNA sequencing alongside data from in-stent restenosis patients and various experimental in-stent restenosis models (swine, rats, and mice), we investigated the role of PFN1 in promoting VSMC phenotype switching and neointimal hyperplasia. RESULTS: Single-cell RNA sequencing of stenotic rat carotid arteries revealed a critical role for PFN1 in neointimal hyperplasia, a finding corroborated in stented swine coronary arteries, in-stent restenosis patients, PFN1 ^SMC-IKO (SMC-specific PFN1 knockout) mice, and PFN1 overexpressed mice. PFN1 deletion was shown to suppress VSMC phenotype switching and neointimal hyperplasia in PFN1 ^SMC-IKO mice subjected to a wire-injured model. To elucidate the observed discordance in PFN1 mRNA and protein levels, we identified that METTL3 (N ⁶ -methyladenosine methyltransferase) and YTHDF3 (YTH N6-methyladenosine RNA binding protein F3; N ⁶ -methyladenosine–specific reader) enhance PFN1 translation efficiency in an N ⁶ -methyladenosine–dependent manner, confirmed through experiments involving METTL3 knockout and YTHDF3 knockout mice. Furthermore, PFN1 was mechanistically found to interact with the phosphorylation of ANXA2 (annexin A2) by recruiting Src (SRC proto-oncogene, nonreceptor tyrosine kinase), promoting the phosphorylation of STAT3 (signal transducer and activator of transcription 3), a typical transcription factor known to induce VSMC phenotype switching. CONCLUSIONS: This study unveils the significance of PFN1 N ⁶ -methyladenosine modification in VSMCs, demonstrating its role in promoting phenotype switching and neointimal hyperplasia through the activation of the p-ANXA2 (phospho-ANXA2)/STAT3 pathway.

Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by excessive proliferation and vasoconstriction of small pulmonary artery vascular smooth muscle cells (PASMCs). Coptidis rhizoma (CR) because of the complexity of the components, the underlying pharmacological role and mechanism of it on PAH remains unknown. In this article, the network pharmacological analysis was used to screen the main active constituents of CR and the molecular targets that these constituents act on. Then, we evaluated the importance of berberine and quercetin (biologically active components of CR) on the proliferation and migration of PASMCs and vascular remodeling in experimental models of PAH. Our results showed that berberine and quercetin effectively inhibited the proliferation and migration of hypoxia-induced PASMCs in a manner likely to be mediated by the suppression of MAPK1, NADPH oxidase 4 (NOX4), and cytochrome P450 1B1 (CYP1B1) expression. Furthermore, berberine and quercetin treatment attenuates pulmonary hypertension, reduces right ventricular hypertrophy, and improves pulmonary artery remodeling in monocrotaline-induced pulmonary hypertension in rat models. In conclusion, this research demonstrates CR might be a promising treatment option for PAH, and the network pharmacology approach can be an effective tool to reveal the potential mechanisms of Chinese herbal medicine.