Inhibiting vascular smooth muscle cell proliferation mediated by osteopontin via regulating gut microbial lipopolysaccharide: A novel mechanism for paeonol in atherosclerosis treatment

Abstract

Background: Although the gut microbiota is involved in metabolic disease such as atherosclerosis, the underlying mechanism remains elusive. Paeonol (Pae) is a natural phenolic compound isolated from Cortex Moutan, which exhibits anti-atherosclerotic effects. Our previous research demonstrated gut microbiota as a site of Pae action. However, the mechanism by which Pae exerts its anti-atherosclerotic effect by the regulation of gut microbiota remains unclear.Objective: To investigate a potential mechanistic link between the gut microbial lipopolysaccharide (LPS) and vascular smooth muscle cell (VSMC) proliferation in atherosclerosis progression and explore the possible role of Pae.Methods: Experimental atherosclerosis was established in ApoE−/− mice, and the atherosclerosis mice were treated with Pae for 4 weeks before being sacrificed for analyses while conducting fecal microbiota transplantation (FMT). The plaque area, levels of serum LPS, expressions of inflammatory factors in serum or aorta, and intestinal barrier permeability were determined. VSMCs were co-cultured with THP-1 cells. CCK-8 assay and EdU staining were performed to assess the proliferative capacity of VSMCs. Immunofluorescence staining was performed to observe the nuclear transfer of p65. Western blotting was used to detect the candidate protein expression level, and quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression level in tissues or cells of each group.Results: During atherosclerosis progression, gut dysbiosis leads to the peripheral accumulation of gut microbial LPS, which acts as a trigger to stimulate osteopontin (OPN) production from circulating monocytes, inducing cell-to-cell crosstalk to promote VSMC proliferation in the aorta. Importantly, the elevation of LPS and OPN concentrations in the blood was also observed in patients with atherosclerosis. Pae could significantly improve atherosclerosis, suppress gut microbial LPS accumulation, and inhibit monocyte/macrophage activation and VSMC proliferation.Conclusions: The present study provides a mechanistic scenario for how long-term stimulation of gut microbial LPS in circulating blood generates a pathological secondary response that leads to abnormal proliferation of VSMCs using high OPN expression in circulating monocytes and suggests a novel strategy for atherosclerosis therapy by remodeling the gut microbiota.

Immunoregulatory Roles of Osteopontin in Diseases

“…Resulting from alternative splicing and post-translational modifications, OPN has various isoforms that engage in multiple signaling pathways. The binding of OPN with integrins initiates the activation of several downstream signaling effectors, such as phosphatidylinositol 3 kinase (PI3K) /protein kinase B (AKT), focal adhesion kinase (FAK)/AKT and nuclear factor kappa-B (NF-κB), leading to cell proliferation, migration, epithelial–mesenchymal transition (EMT), inflammation, neurotoxic microglial phenotype, tumor growth, migration and invasion, as well as angiogenesis within the chronic subdural hematoma (CSDH) outer membrane [ 15 , 16 , 17 , 18 , 19 , 20 ]. In addition, OPN regulates other signal pathways or signal molecules, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT) [ 21 , 22 , 23 , 24 ], PI3K/AKT [ 15 , 25 , 26 , 27 ], NOTCH [ 28 , 29 ], extracellular regulated protein kinase1/2 (ERK1/2) [ 30 ], the ubiquitin C -terminal hydrolase L1 (UCHL1)–ubiquitin–proteasome system (UPS) axis [ 31 ] and transforming growth factor β (TGF-β) [ 32 ], influencing cellular physiological processes and disease progression.…”

“…In atherosclerosis, gut dysbiosis increases the production of gut microbial lipopolysaccharide (LPS), which stimulates OPN expression in circulating monocytes and promotes vascular smooth muscle cell (VSMC) proliferation via the αvβ3/NF-κB pathway. Paeonol (Pae), a natural phenolic compound, inhibits atherosclerosis by reducing gut microbial LPS and OPN levels and blocking the crosstalk between monocytes and VSMCs [ 16 ]. Furthermore, OPN levels can strongly predict clinical outcome of stable patients with chronic heart failure [ 77 ].…”

Engineering molecular nanoprobes to target early atherosclerosis: Precise diagnostic tools and promising therapeutic carriers

“…Therefore, this bimodal imaging can offer a more precise mode for vulnerable plaques, which has been reported to coordinate with TPZ/IR780@HSAeOPN nanoprobes or Fe 3 O 4 nanoparticle-based probes, and introduced in this subchapter as well 27 , 111 . Both nanoprobes target osteopontin, which shows a high expression pattern in plaques, and is closely associated with smooth muscle cell proliferation and foamy macrophage formation 112 , 113 . Based on this rationale, Xu together with colleagues established osteopontin-targeted theranostic nanoprobes, which can precisely regress VASPs through a cascade of synergistic events triggered by local irradiation of lasers under the guidance of fluorescence/MR imaging.…”

Gut Molecules in Cardiometabolic Diseases: The Mechanisms behind the Story

“…In an atherosclerosis-prone animal model, increased LPS levels, as a marker of gut dysbiosis, potentiated the progression of atherosclerotic plaques and promoted the proliferation of vascular smooth muscle cells via osteopontin production by monocytes in an NF-κB dependent manner. Moreover, in patients with carotid atherosclerosis, LPS and osteopontin levels were increased and showed a positive correlation, indicating a relationship between gut dysbiosis and atherogenesis both in humans and animals [ 142 ]. In patients with PAD, increased LPS levels were associated with the atherosclerotic burden and markers of oxidative stress activation [ 143 ].…”