OGT deficiency in vascular smooth muscle orchestrates foam cell formation and PANoptosis during atherosclerotic progression.

The role that vascular smooth muscle cell (VSMC)-derived foam cells play as drivers of atherosclerosis has been an increasing focus of recent research interest. Toll-like receptor 4 (TLR4) has been identified as a regulator of the formation of VSMC foam cells, while vitamin D can reportedly suppress macrophage-derived foam cell development. Our aim is to investigate whether vitamin D can similarly suppress the formation of VSMC foam cells, as well as the role of TLR4 in this pathogenic context. The impact of vitamin D on VSMC-derived foam cell and atherosclerotic plaque formation was assessed, and the expression of cholesterol transport-related genes and TLR4 was assessed in ApoE-/- mice. The impact of 1,25(OH)2D3 on the ox-LDL-mediated formation of foam cells and the underlying molecular mechanisms were also examined in VSMCs cultured in vitro. Supplemental vitamin D administration resulted in a pronounced reduction in aortic atherosclerotic plaque formation and the development of SMA-α-positive foam cells. Vitamin D further suppressed TLR4, CD36 and SR-A in atherosclerotic plaque lesions while promoting ABCA1, ABCG1 and LXR-α upregulation. 1,25(OH)2D3 significantly reduced Dil-ox-LDL uptake and increased NBD-LDL efflux in VSMCs, in addition to suppressing TLR4, CD36 and SR-A expression, while upregulating ABCA1, ABCG1 and LXR-α. TLR4 knockdown impaired VSMC foam cell formation, while 1,25(OH)2D3-induced JNK activation suppressed TLR4 signaling and promoted VSMC foam cell development. Our study reveals that vitamin D can reduce VSMC foam cell formation and protect against atherosclerotic progression through the JNK-TLR4 signaling pathway.

Huanglian Jiedu decoction inhibits vascular smooth muscle cell-derived foam cell formation by activating autophagy via suppressing P2RY12

Increasing evidence suggests that exposure of particulate matter (PM) from traffic vehicles, e.g., diesel exhaust particles (DEP), was associated with adverse vascular effects, e.g., acceleration of atherosclerotic plaque progression. By analogy, engineered nanoparticles (NPs) could also induce similar effects. The formation of lipid laden foam cells, derived predominately from macrophages and vascular smooth muscle cells (VSMC), is closely associated with the development of atherosclerosis and adverse vascular effects. We reviewed current studies about particle exposure-induced lipid laden foam cell formation. In vivo studies using animal models have shown that exposure of air pollution by PM promoted lipid accumulation in alveolar macrophages or foam cells in plaques, which was likely associated with pulmonary inflammation or systemic oxidative stress, but not blood lipid profile. In support of these findings, in vitro studies showed that direct exposure of cultured macrophages to DEP or NP exposure, with or without further exposure to external lipids, promoted intracellular lipid accumulation. The mechanisms remained unknown. Although a number studies found increased reactive oxygen species (ROS) or an adaptive response to oxidative stress, the exact role of oxidative stress in mediating particle-induced foam cell formation requires future research. There is currently lack of reports concerning VSMC as a source for foam cells induced by particle exposure. In the future, it is necessary to explore the role of foam cell formation in particle exposure-induced atherosclerosis development. In addition, the formation of VSMC derived foam cells by particle exposure may also need extensive studies.

Oxidized low-density lipoprotein (oxLDL) induced a foam-cell-like phenotype of the vascular smooth muscle cells (VSMCs), leading to the inflammatory responses incorporating Toll-like receptor- (Tlr-) mediated cellular alterations. However, the role of Tlr4 in foam cell formation and underlying molecular pathways has not been comprehensively elucidated. To further investigate the mechanism, VSMCs were incubated with different doses of oxLDL, and then, the lipid, reactive oxygen species (ROS) accumulation, Tlr family genes, and the foam cell phenotype were explored. We observed that oxLDL induced foam cell-like phenotype in VSMCs and led to lipid and ROS accumulation in a dose-dependent manner. Furthermore, in the Tlr family, Tlr4 demonstrated the strongest upregulation under oxLDL stimulation. Simultaneously, oxLDL induced activation of Src, higher expression of Nox2, and lower expression of Mnsod, Sirt1, and Sirt3. By interfering the TLR4 expression, the phenotype alteration, lipid accumulation in VSMCs, and Src kinase activation induced by oxLDL were abolished. After interfering Src activation, the oxLDL-induced lipid accumulation and foam cell phenotype in VSMCs were also alleviated. Furthermore, the ROS accumulation, upregulated Nox2 expression, downregulated Sirt1, Sirt3, and Mnsod expression in VSMCs under oxLDL stimulation were also relieved after the knockdown of Tlr4. Additionally, overexpression of Sirt1 and Sirt3 ameliorated the ROS accumulation and foam cell-like marker expression in VSMCs. These results demonstrated that beyond its familiar role in regulating inflammation response, Tlr4 is a critical regulator in oxLDL-induced foam cell formation in VSMCs via regulating Src kinase activation as well as Sirt1 and Sirt3 expression.

Homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS). Hcy induces the transformation of vascular smooth muscle cells (VSMCs) into foam cells, which play a crucial role in this process. However, the detailed mechanism is still unclear. To identify the key regulatory proteins during this process and clarify the possible mechanism of Hcy-induced foam cell formation in VSMCs, thereby providing theoretical support for the intervention of AS. VSMCs were allocated into two groups: a control cohort and a group exposed to Hcy to simulate an AS-like state. Quantitative proteomic profiling was performed using the label-free quantitative DIA (LFQ-DIA) approach to detect differentially expressed proteins between these groups. To explore functional implications, enrichment analyses involving Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were conducted. Protein-protein interaction networks were constructed using the STRING database to identify central interactors. Target proteins were subsequently validated through parallel reaction monitoring (PRM). Furthermore, histological analyses (hematoxylin and eosin (HE) staining, Oil Red O staining), biochemical assays of lipid content (total cholesterol (TC) and triglycerides (TG)), and Western blot analysis were utilized to confirm the role and mechanism of identified proteins in the context of Hcy-driven foam cell conversion. The results showed that proteomic analysis identified 4804 proteins in total, of which 4799 passed missing-value filtering and were retained for downstream quantitative analysis. A total of 54 proteins were identified as differentially expressed using thresholds of adjusted p-value < 0.05 and fold change > 1.5. Among them, 13 proteins were upregulated, while 41 were downregulated in response to Hcy treatment. For PRM validation, 20 candidate proteins were selected according to proteomic evidence, biological relevance, and technical feasibility. Among them, 16 proteins (COX7C, STX5, UBQLN2, DDX50, TBCB, GSR, PCNP, CDV3, PEBP1, PPIA, S100A6, EIF4E2, UBQLN1, ARMC1, NUDCD2, and H1-2) showed the same direction of fold-change values as in the LFQ-DIA dataset, thereby underscoring the reliability of the proteomic analysis. Data are available via ProteomeXchange with identifier PXD064315. Histological staining demonstrated enhanced lipid accumulation, and the protein expression of the contraction phenotype marker a-SMA decreased, while the protein expression of the synthesis phenotype marker OPN increased. This indicates that Hcy induces VSMCs to transform from a contraction phenotype to a synthesis phenotype, resulting in the formation of foam cells. The protein levels of COX7C and sterol regulatory element-binding proteins (SREBP1C and SREBP2) were elevated upon Hcy exposure. Overexpression of COX7C further augmented the expression of SREBP1C and SREBP2, exacerbated lipid accumulation, and promoted foam cell transformation in Hcy-treated VSMCs. On the other hand, knockdown of COX7C had the opposite effect. Overall, the results of the present study suggest that COX7C plays a crucial regulatory role in Hcy-induced transformation of VSMCs into foam cells. Its pathogenic role is likely mediated through the upregulation of SREBP1C and SREBP2, thereby promoting lipid accumulation. These findings provide new insights into AS pathogenesis and identify COX7C maybe a potential therapeutic target.

A defining feature of atherosclerosis is the uptake of oxidized lipids by cells, leading them to differentiate into foam cells. Foam cells were once believed to originate primarily from macrophages. However, recent research suggests that over 70% are derived from vascular smooth muscle cells (VSMCs). While the ability of VSMCs to undergo phenotypic modulation into foam cells is established, the transcriptomic distinctions and regulatory mechanisms regulating this transition remain poorly characterized. We hypothesized that given their phenotypic and functional differences; VSMC-derived foam cells would have a distinct gene expression and regulatory signature compared to contractile VSMC. To investigate this, we conducted bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing LDLR -/- mouse models maintained on a high-fat diet. Using LipidTox staining VSMC and VSMC-derived foam cells were identified, and isolated using flow cytometry at key time points representing late and advanced stages of disease progression. Computational analyses revealed distinct differentially expressed genes, key molecular regulatory functions, and enriched biological pathways, including pathways such as cell cycle signalling, integrin interactions and interleukin signalling, that differentiate VSMC-derived foam cells from their contractile counterparts. Our findings also reveal that the expression of the Basic Helix-Loop-Helix Family Member E40, Bhlhe40 (also known as Dec1), a pro-inflammatory transcription factor, is activated in VSMC-derived foam cells during advanced stages of atherosclerosis and enriched in modulated and proliferative VSMC-derived cells measured at scRNA-seq of atherosclerotic lesions. Upstream regulator analysis suggests that Bhlhe40 may play a pivotal role in driving the differentiation of VSMCs into foam cells. Functional experiments revealed that Bhlhe40 knockdown in vitro inhibits VSMC phenotypic modulation and lipid uptake in response to cotreatment withTNFα and MBD-Cholesterol for 48 hours which promotes foam cell formation. These findings reveal a modulatory role for Bhlhe40 in the VSMC response to atherosclerotic stress, positioning it as a potential therapeutic target for regulating VSMC-derived foam cell formation in atherosclerosis.

Accumulation of lipids in in the arterial wall is a key feature of atherosclerosis. In atherosclerotic plaque from human and mouse origin it has recently become appreciated that more than 50% of total foam cells are derived from vascular smooth muscle cells (VSMCs) suggesting a much larger role for VSMCs in foam cell formation then previously assumed. The molecular mechanism behind this process and clinical significance remains yet to be elucidated. Myocardin Related Transcription Factor –A (MRTFA) is a transcriptional co‐activator that has been demonstrated to play a key role in pathological vascular remodelling including progression of atherosclerotic lesions. Here we aim to investigate the functional role of MRTFA on cholesterol loading of VSMCs.To address this, human coronary artery smooth muscle cells (HCASMCs) were loaded with cholesterol‐cyclodextrin complexes for 96 hours. Foam cell formation was evident by accumulation of intracellular Oil red O‐stained lipids. Notably, overexpression of MRTFA increased lipid accumulation with considerable build‐up of lipid droplets in the cytoplasm. Small molecule N‐cyclopropyl‐5‐(thiophen‐2‐yl)‐isoxazole‐3‐carboxamide (ISX), an activator of MRTFA‐driven transcription, further potentiated this effect. Although expression of macrophage markers by mouse SMCs following cholesterol loading has been demonstrated, we did not observe any consistent change in the expression of macrophage or SMCs markers in lipid loaded cells of human origin.These preliminary findings suggest that MRTFA in VSMCs plays a significant role in foam cell formation following cholesterol loading. A better understanding of the mechanisms for cholesterol handling may enable us to come up with better therapeutic strategies for prevention of cholesterol accumulation in the arterial walls and its clinical outcomes.Support or Funding InformationThis work was supported by the Swedish Research Council; the Swedish Heart and Lung Foundation, the Novo Nordisk Foundation, the Crafoord Foundation; the Royal Physiographic Society and the Magnus Bergvall Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Foam cell formation is the hallmark of atherosclerosis. Both telmisartan and autophagy protect against the development of atherosclerosis. However, it has yet to be elucidated whether telmisartan prevents vascular smooth muscle cell (VSMC)-derived foam cell formation. Vascular smooth muscle cells isolated from the thoracic aorta of male C57BL/6J mice were used for this study. To induce foam cell formation, primary VSMCs were incubated in 80 μg/ml oxLDL for 24 h. LC3, beclin-1, PPARγ, AMPK, p-AMPK, mTOR and p-mTOR expression were determined via Western blot. Lipid accumulation was evaluated via oil red O staining and intracellular total cholesterol level measurement. Our study demonstrated that telmisartan dose-dependently increased the expression of beclin-1, the LC3II/LC3I ratio and the quantity of GFP-labeled autophagosomes, displaying a peak effect at 10 μM. In control siRNA-transfected VSMCs, telmisartan (10 μM) decreased lipid droplet accumulation and the total cholesterol level significantly. In contrast, in Atg7 siRNA-transfected VSMCs, telmisartan failed to attenuate lipid accumulation. In addition, telmisartan dose-dependently increased the expression of PPARγ and p-AMPK and decreased the expression of p-mTOR. GW9662 attenuated the telmisartan-induced increase in PPARγ expression, the LC3-II/LC3-I ratio and p-AMPK expression and the telmisartan-induced decrease in p-mTOR expression. Compound C restored mTOR activity and abolished the increase in the LC3-II/LC3-I ratio. Rapamycin significantly reduced p-mTOR expression and increased the LC3-II/LC3-I ratio. In conclusion, this study provides evidence that the chronic pharmacological activation of the PPARγ-mediated autophagy pathway using telmisartan may represent a promising therapeutic strategy for atherosclerosis.

BackgroundOxidized low-density lipoproteins (ox-LDL) may induce foam cell formation from the vascular smooth muscle cell (VSMC) by inhibiting VSMC autophagy. This process accelerates the formation of atherosclerosis (AS). Connexin 43 (Cx43), which is the most widely distributed connexin in VSMC is associated with autophagy. However, the mechanism of action and the involvement of Cx43 in ox-LDL-inhibited VSMC autophagy remain unclear.MethodsThe primary VSMC were obtained and identified, before primary VSMC were pretreated with an inhibitor (Cx43-specific inhibitor Gap26 and PI3K inhibitor LY294002) and stimulated with ox-LDL.ResultsOx-LDL not only inhibited autophagy in VSMC via downregulation of autophagy-related proteins (such as Beclin 1, LC3B, p62), but also increased Cx43 protein levels. Then we added Gap26 to VSMC in the ox-LDL+Gap26 group, in which autophagy-related proteins were increased and the accumulation of lipid droplets was reduced. These result suggested that an enhanced level of autophagy and an alleviation of lipid accumulation might be caused by inhibiting Cx43 in VSMC. The phosphorylation levels of PI3K, AKT, mTOR were increased by ox-LDL, thus down-regulating autophagy-related proteins. However, this situation was partially reversed by the Gap26. Moreover, Cx43 expression were decreased by LY294002 in ox-LDL-induced VSMCs.ConclusionInhibiting Cx43 may activate VSMC autophagy to inhibit foam cell formation by inhibiting the PI3K/AKT/mTOR signaling pathway.

The aim of this work was to elucidate the effect of cyclic nucleotide signaling on the growth of vascular smooth muscle cells (VSMCs). In particular, the role of cGKI in VSMC growth was analysed in primary and subcultured VSMCs derived from wild-type and cGKI-deficient mice. In primary VSMCs, activation of cGMP/cGKI signaling led to a strong increase in growth. In contrast, in repeatedly passaged VSMCs derived from mouse, rat and human, cGMP/cGKI had either no effect on growth or had a weak growth suppressing effect. Thus, cGKI signaling differs in primary vs. subcultured VSMCs. The further analysis of proliferation, apoptosis, cytoskeletal dynamics, and various signaling pathways indicated that an increase in integrin-mediated cell adhesion is the major mechanism for cGKI-mediated growth in primary VSMCs. Thereby, cGMP/cGKI signaling in primary VSMCs might inhibit anoikis, the programmed cell death induced by the loss of cell/matrix interactions.

Homeobox A9 is a novel mediator of vascular smooth muscle cell phenotypic switching and proliferation by regulating methyl-CpG binding protein 2

BackgroundThis study aimed at investigating whether NLRP3 (the Nod like receptor family, pyrin domain‐containing 3 protein) inflammasome activation induced HMGB1 (high mobility group box‐1 protein) secretion and foam cell formation in human vascular smooth muscle cells (VSMCs) and atherosclerosis in ApoE−/− mice.Methods and Results VSMCs or ApoE−/− mice were treated with lipopolysaccharides (LPS) and/or ATP or LPS and high‐fat diet to induce NLRP3 inflammasome activation. HMGB1 distribution and foam cell formation in VSMCs were characterized. Liver X receptor α and ATP‐binding cassette transporter expression were determined. The impact of NLRP3 or receptor for advanced glycation end product silencing, ZYVAD‐FMK (caspase‐1 inhibitor), glycyrrhizin (HMGB1 inhibitor) or receptor for advanced glycation end product antagonist peptide on HMGB1 secretion, foam cell formation, liver X receptor α and ATP‐binding cassette transporter expression was examined. Expression level of HMGB1 in human atherosclerosis obliterans arterial tissues was characterized. Our results found that NLRP3 inflammasome activation promoted foam cell formation and HMGB1 secretion in VSMCs. Extracellular HMGB1 was a key signal molecule in inflammasome activation‐mediated foam cell formation. Furthermore, inflammasome activation‐induced HMGB1 activity and foam cell formation were achieved by receptor for advanced glycation end product/liver X receptor α /ATP‐binding cassette transporter glycyrrhizin. Experiments in vivo found glycyrrhizin significantly attenuated the LPS/high‐fat diet‐induced atherosclerosis and serum HMGB1 levels in mice. Finally, levels of HMGB1 and NLRP3 were increased in tunica media adjacent to intima of atherosclerosis obliteran arteries.ConclusionsOur results revealed that HMGB1 is a key downstream signal molecule of NLRP3 inflammasome activation and plays an important role in VSMCs foam cell formation and atherogenesis by downregulating liver X receptor α and ATP‐binding cassette transporter expression through receptor for advanced glycation end product.

Nesfatin-1 functions as a switch for phenotype transformation and proliferation of VSMCs in hypertensive vascular remodeling

Phthalate promotes atherosclerosis through interacting with long-non coding RNA and induces macrophage foam cell formation and vascular smooth muscle damage

Geniposide ameliorates atherosclerosis by restoring lipophagy via suppressing PARP1/PI3K/AKT signaling pathway