Statin suppresses the development of excessive intimal proliferation in a Kawasaki disease mouse model.

Kawasaki disease (KD) causes vascular injury and lifelong remodeling. Excessive intimal proliferation has been observed, resulting in coronary artery lesions (CALs). However, the mechanisms underlying vascular remodeling in CAL and statin treatment have not been comprehensively elucidated. This study aimed to investigate the effects of statins on vascular remodeling using a KD mouse model. Candida albicans water-soluble substance (CAWS) was intraperitoneally injected in 5-week-old male apolipoprotein-E-deficient mice. They were categorized as follows (n = 4): control, CAWS, CAWS+statin, and late-statin groups. The mice were euthanized at 6 or 10 weeks after injection. Statins (atorvastatin) were initiated after CAWS injection, except for the late-statin group, for which statins were internally administered 6 weeks after injection. Elastica van Gieson staining and immunostaining were performed for evaluation. Statins substantially suppressed the marked neointimal hyperplasia induced by CAWS. Additionally, CAWS induced TGFß receptor II and MAC-2 expression around the coronary arteries, which was suppressed by the statins. KD-like vasculitis might promote the formation of aneurysm by destroying elastic laminae and inducing vascular stenosis by neointimal proliferation. The anti-inflammatory effects of statins might inhibit neointimal proliferation. Therefore, statin therapy might be effective in adult patients with KD with CAL by inhibiting vascular remodeling.

Restoration of ARA metabolic disorders in vascular smooth muscle cells alleviates intimal hyperplasia.

Intimal hyperplasia (IH) is an innegligible issue for patients undergoing interventional therapy. The proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor-BB (PDGF-BB) are critical events in the development of IH. While the exact mechanism and effective target for IH needs further investigation. Metabolic disorders of arachidonic acid (ARA) are involved in the occurrence and progression of various diseases. In this study, we found that the expressions of soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) were significantly increased in the VSMCs during balloon injury-induced IH. Then, we employed a COX-2/sEH dual inhibitor PTUPB to increase the concentration of epoxyeicosatrienoic acids (EETs) while prevent the release of pro-inflammatory prostaglandins. Results showed that PTUPB treatment significantly reduced neointimal thickening induced by balloon injury in rats in vivo and inhibited PDGF-BB-induced proliferation and migration of VSMCs in vitro. Our results showed that PTUPB may reverse the phenotypic transition of VSMCs by inhibiting Pttg1 expression. In conclusion, we found that the dysfunction of ARA metabolism in VSMCs contributes to IH, and the COX-2/sEH dual inhibitor PTUPB attenuates IH progression by reversing the phenotypic switch in VSMC through the Sirt1/Pttg1 pathway.

Targeting Skp2 degradation with troxerutin decreases neointima formation.

The proliferative and migratory abilities of vascular smooth muscle cells (VSMCs) play a crucial role in neointima formation following vascular injury. Skp2 facilitates proliferation and migration in cells through cell cycle regulation, presenting an important therapeutic target for atherosclerosis, pulmonary hypertension, and vascular restenosis. This study aimed to identify a natural product capable of inhibiting neointima formation post vascular injury. Here, we demonstrate that troxerutin, a flavonoid, significantly reduced viability and downregulated Skp2 in VSMCs. Moreover, troxerutin exhibited anti-proliferative effects on VSMCs and mitigated neointima formation. These findings collectively elucidate the intrinsic mechanism of troxerutin in treating atherosclerosis, pulmonary hypertension, and vascular restenosis by targeting the E3-linked enzyme Skp2.

Cucurbitacins mitigate vascular neointimal hyperplasia by suppressing cyclin A2 expression and inhibiting VSMC proliferation.

BACKGROUND: Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health. Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug-eluting stent is an effective approach to improve restenosis. Cucurbitacins have been demonstrated to exert an anti-proliferation effect in various tumors and a hypotensive effect. This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L. (CuECs) and cucurbitacin B (CuB) on restenosis. METHODS: C57BL/6 mice were subjected to left carotid artery ligation and subcutaneously injected with CuECs or CuB for 4 weeks. Hematoxylin-Eosin, immunofluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia. Western blot, real-time PCR, flow cytometry analysis, EdU staining and cellular immunofluorescence assay were employed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro. The potential interactions of CuECs with cyclin A2 were performed by molecular docking. RESULTS: The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells. Furthermore, CuECs and CuB mediated cell cycle arrest at the S phase. Autodocking analysis demonstrated that CuB, CuD, CuE and CuI had high binding energy for cyclin A2. Our study also showed that CuECs and CuB dramatically inhibited FBS-induced cyclin A2 expression. Moreover, the expression of cyclin A2 in CuEC- and CuB-treated neointima was downregulated. CONCLUSIONS: CuECs, especially CuB, exert an anti-proliferation effect in VSMCs and may be potential drugs to prevent restenosis.

Kanglexin counters vascular smooth muscle cell dedifferentiation and associated arteriosclerosis through inhibiting PDGFR.

BACKGROUND: Dysregulation of vascular smooth muscle cell (VSMC) function leads to a variety of diseases such as atherosclerosis and hyperplasia after injury. However, antiproliferative drug targeting VSMC exhibits poor specificity. Therefore, there is an urgent to develop highly specific antiproliferative drugs to prevention and treatment VSMC dedifferentiation associated arteriosclerosis. Kanglexin (KLX), a new anthraquinone compound designed by our team, has potential to regulate VSMC phenotype according to the physicochemical properties. PURPOSE: This project aims to evaluate the therapeutic role of KLX in VSMC dedifferentiation and atherosclerosis, neointimal formation and illustrates the underlying molecular mechanism. METHODS: In vivo, the ApoE-/- mice were fed with high-fat diet (HFD) for a duration of 13 weeks to establish the atherosclerotic model. And rat carotid artery injury model was performed to establish the neointimal formation model. In vitro, PDGF-BB was used to induce VSMC dedifferentiation. RESULTS: We found that KLX ameliorated the atherosclerotic progression including atherosclerotic lesion formation, lipid deposition and collagen deposition in aorta and aortic sinus in atherosclerotic mouse model. In addition, The administration of KLX effectively ameliorated neointimal formation in the carotid artery following balloon injury in SD rats. The findings derived from molecular docking and surface plasmon resonance (SPR) experiments unequivocally demonstrate that KLX had potential to bind PDGFR-ß. Mechanism research work proved that KLX prevented VSMC proliferation, migration and dedifferentiation via activating the PDGFR-ß-MEK -ERK-ELK-1/KLF4 signaling pathway. CONCLUSION: Collectively, we demonstrated that KLX effectively attenuated the progression of atherosclerosis in ApoE-/- mice and carotid arterial neointimal formation in SD rats by inhibiting VSMC phenotypic conversion via PDGFR-ß-MEK-ERK-ELK-1/KLF4 signaling. KLX exhibits promising potential as a viable therapeutic agent for the treatment of VSMC phenotype conversion associated arteriosclerosis.

Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

Bergenin alleviates proliferative arterial diseases by modulating glucose metabolism in vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation and phenotypic switching are key mechanisms in the development of proliferative arterial diseases. Notably, reprogramming of the glucose metabolism pattern in VSMCs plays an important role in this process. PURPOSE: The aim of this study is to investigate the therapeutic potential and the mechanism underlying the effect of bergenin, an active compound found in Bergenia, in proliferative arterial diseases. METHODS: The effect of bergenin on proliferative arterial disease was evaluated using platelet-derived growth factor (PDGF)-stimulated VSMCs and a mouse model of carotid artery ligation. VSMC proliferation and phenotypic switching were evaluated in vitro using cell counting kit-8, 5-ethynyl-2-deoxyuridine incorporation, scratch, and transwell assays. Carotid artery neointimal hyperplasia was evaluated in vivo using hematoxylin and eosin staining and immunofluorescence. The expression of proliferation and VSMC contractile phenotype markers was evaluated using PCR and western blotting. RESULTS: Bergenin treatment inhibited PDGF-induced VSMC proliferation and phenotypic switching and reduced neointimal hyperplasia in the carotid artery ligation model. Additionally, bergenin partially reversed the PDGF-induced Warburg-like glucose metabolism pattern in VSMCs. RNA-sequencing data revealed that bergenin treatment significantly upregulated Ndufs2, an essential subunit of mitochondrial complex I. Ndufs2 knockdown attenuated the inhibitory effect of bergenin on PDGF-induced VSMC proliferation and phenotypic switching, and suppressed neointimal hyperplasia in vivo. Conversely, Ndufs2 overexpression enhanced the protective effect of bergenin. Moreover, Ndufs2 knockdown abrogated the effects of bergenin on the regulation of glucose metabolism in VSMCs. CONCLUSION: These findings suggest that bergenin is effective in alleviating proliferative arterial diseases. The reversal of the Warburg-like glucose metabolism pattern in VSMCs during proliferation and phenotypic switching may underlie this therapeutic mechanism.

Isoxanthohumol reduces neointimal hyperplasia through the apelin/AKT pathway.

The abnormal proliferation, migration, and inflammation of vascular smooth muscle cells (VSMCs) play crucial roles in the development of neointimal hyperplasia and restenosis. Exposure to inflammatory cytokines such as platelet-derived growth factor (PDGF)-BB and tumour necrosis factor-alpha (TNF-α) induces the transformation of contractile VSMCs into abnormal synthetic VSMCs. Isoxanthohumol (IXN) has significant anti-inflammatory, antiproliferative, and antimigratory effects. This study aimed to explore the therapeutic impact and regulatory mechanism of IXN in treating neointimal hyperplasia. The present findings indicate that IXN effectively hinders the abnormal proliferation, migration, and inflammation of VSMCs triggered by PDGF or TNF-α. This inhibition is primarily achieved through the modulation of the apelin/AKT or AKT pathway, respectively. In an in vivo model, IXN effectively reduced neointimal hyperplasia in denuded femoral arteries. These results suggest that IXN holds promise as a potential and innovative therapeutic candidate for the treatment of restenosis.

Myeloid-derived growth factor suppresses VSMC dedifferentiation and attenuates postinjury neointimal formation in rats by activating S1PR2 and its downstream signaling.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 µg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature.

Ultrasound-guided periadventitial administration of rapamycin-fibrin glue attenuates neointimal hyperplasia in the rat carotid artery injury model.

INTRODUCTION: Arterial restenosis caused by intimal hyperplasia (IH) is a serious complication after vascular interventions. In the rat carotid balloon injury model, we injected phosphate buffer saline (PBS), rapamycin-phosphate buffer saline suspension (RPM-PBS), blank fibrin glue (FG) and rapamycin-fibrin glue (RPM-FG) around the injured carotid artery under ultrasound guidance and observed the inhibitory effect on IH. METHODS: The properties of RPM-FG in vitro were verified by scanning electron microscopy (SEM) and determination of the drug release rate. FG metabolism in vivo was observed by fluorescence imaging. The rat carotid balloon injury models were randomly classified into 4 groups: PBS group (control group), RPM-PBS group, FG group, and RPM-FG group. Periadventitial administration was performed by ultrasound-guided percutaneous puncture on the first day after angioplasty. Carotid artery specimens were analyzed by immunostaining, Evans blue staining and hematoxylin-eosin staining. RESULTS: The RPM particles showed clustered distributions in the FG block. The glue was maintained for a longer time in vivo (> 14 days) than in vitro (approximately 7 days). Two-component liquid FG administered by ultrasound-guided injection completely encapsulated the injured artery before coagulation. The RPM-FG inhibited IH after carotid angioplasty vs. control and other groups. The proliferation of vascular smooth muscle cells (VSMCs) was significantly inhibited during neointima formation, whereas endothelial cell (EC) repair was not affected. CONCLUSION: Periadventitial delivery of RPM-FG contributed to inhibiting IH in the rat carotid artery injury model without compromising re-endothelialization. Additionally, FG provided a promising platform for the future development of a safe, effective, and minimally invasive perivascular drug delivery method to treat vascular disease.

Mechanistic insights into the anti-restenotic effects of HSP27 and HO1 modulated by reconstituted HDL on neointimal hyperplasia.

High-density lipoprotein (HDL) therapy has demonstrated beneficial effects in acute stroke and acute myocardial infarction models by reducing infarct size. In this study, we investigated the inhibitory effects of reconstituted HDL (rHDL) on neointimal hyperplasia and elucidated its underlying mechanism using a balloon injury rat model. Our finding revealed a significant 37% reduction in the intima to media ratio in the arteries treated with 80 mg/kg rHDL compared to those subjected to injury alone (p < 0.05), indicating a specific inhibition of neointimal hyperplasia. In vivo analysis further supported the positive effects of rHDL by demonstrating a reduction in smooth muscle cell (SMC) proliferation and an increase in endothelial cell (EC) proliferation. Additionally, rHDL treatment led to decreased infiltration of leukocytes and downregulated the expression of matrix metallopeptidase 9 (MMP9) in the neointimal area. Notably, rHDL administration resulted in decreased expression of VCAM1 and HIF1α, alongside increased expression of heme oxygenase 1 (HO1) and heat shock protein 27 (HSP27). Overexpression of HSP27 and HO1 effectively inhibited SMC proliferation. Moreover, rHDL-mediated suppression of injury-induced HIF1α coincided with upregulation of HSP27. Interestingly, HSP27 and HO1 had varying effects on the expression of chemokine receptors and rHDL did not exert significant effect on chemokine receptor expression in THP1 cells. These findings underscore the distinct roles of HSP27 and HO1 as potential regulatory factors in the progression of restenosis. Collectively, our study demonstrates that rHDL exerts a potent anti-neointimal hyperplasia effect by reducing leukocytes infiltration and SMC proliferation while promoting EC proliferation.

3,4-dimethoxychalcone induces autophagy and reduces neointimal hyperplasia and aortic lesions in mouse models of atherosclerosis.

Autophagy inducers can prevent cardiovascular aging and age-associated diseases including atherosclerosis. Therefore, we hypothesized that autophagy-inducing compounds that act on atherosclerosis-relevant cells might have a protective role in the development of atherosclerosis. Here we identified 3,4-dimethoxychalcone (3,4-DC) as an inducer of autophagy in several cell lines from endothelial, myocardial and myeloid/macrophagic origin, as demonstrated by the aggregation of the autophagosome marker GFP-LC3 in the cytoplasm of cells, as well as the downregulation of its nuclear pool indicative of autophagic flux. In this respect, 3,4-DC showed a broader autophagy-inducing activity than another chalcone (4,4- dimethoxychalcone), spermidine and triethylene tetramine. Thus, we characterized the potential antiatherogenic activity of 3,4-DC in two different mouse models, namely, (i) neointima formation with smooth muscle expansion of vein segments grafted to the carotid artery and (ii) genetically predisposed ApoE-/- mice fed an atherogenic diet. In the vein graft model, local application of 3,4-DC was able to maintain the lumen of vessels and to reduce neointima lesions. In the diet-induced model, intraperitoneal injections of 3,4-DC significantly reduced the number of atherosclerotic lesions in the aorta. In conclusion, 3,4-DC stands out as an autophagy inducer with potent antiatherogenic activity.

Ketogenic diet inhibits neointimal hyperplasia by suppressing oxidative stress and inflammation.

OBJECTIVE: Neointimal hyperplasia is the primary mechanism underlying atherosclerosis and restenosis after percutaneous coronary intervention. Ketogenic diet (KD) exerts beneficial effects in various diseases, but whether it could serve as non-drug therapy for neointimal hyperplasia remains unknown. This study aimed to investigate the effect of KD on neointimal hyperplasia and the potential mechanisms. METHODS AND RESULTS: Carotid artery balloon-injury model was employed in adult Sprague-Dawley rats to induce neointimal hyperplasia. Then, animals were subjected to either standard rodent chow or KD. For in-vitro experiment, impacts of ß-hydroxybutyrate (ß-HB), the main mediator of KD effects, on platelet-derived growth factor BB (PDGF-BB) induced vascular smooth muscle cell (VSMC) migration and proliferation were determined. Balloon injury induced event intimal hyperplasia and upregulation of protein expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), and these changes were significantly ameliorated by KD. In addition, ß-HB could markedly inhibit PDGF-BB induced VMSC migration and proliferation, as well as inhibiting expressions of PCNA and α-SMC. Furthermore, KD inhibited balloon-injury induced oxidative stress in carotid artery, indicated by reduced ROS level, malondialdehyde (MDA) and myeloperoxidase (MPO) activities, and increased superoxide dismutase (SOD) activity. We also found balloon-injury induced inflammation in carotid artery was suppressed by KD, indicated by decreased expressions of proinflammatory cytokines IL-1ß and TNF-α, and increased expression of anti-inflammatory cytokine IL-10. CONCLUSION: KD attenuates neointimal hyperplasia through suppressing oxidative stress and inflammation to inhibit VSMC proliferation and migration. KD may represent a promising non-drug therapy for neointimal hyperplasia associated diseases.

Neointima abating and endothelium preserving - An adventitia-localized nanoformulation to inhibit the epigenetic writer DOT1L.

Open vascular reconstructions such as bypass are common treatments for cardiovascular disease. Unfortunately, neointimal hyperplasia (IH) follows, leading to treatment failure for which there is no approved therapy. Here we combined the strengths of tailoring nanoplatforms for open vascular reconstructions and targeting new epigenetic mechanisms. We produced adhesive nanoparticles (ahNP) that could be pen-brushed and immobilized on the adventitia to sustainably release pinometostat, an inhibitor drug selective to the epigenetic writer DOT1L that catalyzes histone-3 lysine-79 dimethylation (H3K79me2). This treatment not only reduced IH by 76.8% in injured arteries mimicking open reconstructions in obese Zucker rats with human-like diseases but also avoided the shortcoming of endothelial impairment in IH management. In mechanistic studies, chromatin immunoprecipitation (ChIP) sequencing revealed co-enrichment of the histone mark H3K27ac(acetyl) and its reader BRD4 at the gene of aurora kinase B (AURKB), where H3K79me2 was also enriched as indicated by ChIP-qPCR. Accordingly, DOT1L co-immunoprecipitated with H3K27ac. Furthermore, the known IH driver BRD4 governed the expression of DOT1L which controlled AURKB's protein level, revealing a BRD4- > DOT1L- > AURKB axis. Consistently, AURKB-selective inhibition reduced IH. Thus, this study presents a prototype nanoformulation suited for open vascular reconstructions, and the new insights into chromatin modulators may aid future translational advances.

Empagliflozin inhibits neointimal hyperplasia through attenuating endothelial-to-mesenchymal transition via TAK-1/NF-κB pathway.

OBJECTIVE: To investigate whether empagliflozin could prevent injury-induced vascular neointimal hyperplasia and to further explore its mechanism. METHODS: Male C57BL/6J mice were divided into two groups with or without the empagliflozin treatment, and carotid ligation injury was performed to induce neointimal hyperplasia. The injured carotid arteries were collected for Western blotting (WB), histology and immunofluorescence analysis after four weeks. The inflammatory responses were analyzed by qRT-PCR to detect the inflammatory gene mRNA expression. To further explore its mechanism, HUVECs were treated with TGFß-1 to induce EndMT followed by empagliflozin or vehicle treatment in vitro. A23187 (Calcimycin), an agonist of NF-κB signaling was used in the experiment. RESULTS: The wall thickness and the neointima area was significantly reduced in the empagliflozin treatment group on day 28 after artery ligation. The Ki-67 positive cells were 28.33 ± 12.66% and 48.83 ± 10.41% in the empagliflozin-treated group and control group, respectively (P < 0.05). The mRNA expression levels of the inflammatory genes and inflammatory cells were decreased in the empagliflozin treatment group, as well as the MMP2 and MMP9. Meanwhile, empagliflozin can significantly reduce the migratory ability of inflammatory-treated HUVECs. The CD31 was increased in the TGFß1+empagliflozin group, whereas the FSP-1, phosphorylation of TAK-1 (p-TAK-1) and phosphorylation of NF-κB (p- NF-κB) expression level were decreased, compared to the control group without empagliflozin treatment. However, the expression level of FSP-1 and p-NF-κB were reversed after co-treatment with A23187, whereas the p-TAK-1 expression level was without any significant difference. CONCLUSION: Empagliflozin inhibits the inflammation-induced EndMT via the TAK-1/NF-κB signaling pathway.

The innate immune sensor STING accelerates neointima formation via NF-κB signaling pathway.

Vascular smooth muscle cells (VSMCs) proliferation, migration, and phenotypic switching are considered crucial events in the progression of neointima formation. Stimulator of interferon genes (STING), an innate immune sensor of cyclic dinucleotides against pathogens, in neointima formation remains obscure. Here, we observed a significant increase in STING expression on the neointima of injured vessels and mouse aortic VSMCs induced by PDGF-BB. In vivo, global knockout of STING (Sting-/-) attenuated neointima formation after vascular injury. In vitro data showed that STING deficiency significantly alleviated PDGF-BB-induced proliferation and migration in VSMCs. Furthermore, these contractile marker genes were upregulated in Sting-/- VSMCs. Overexpression of STING promoted proliferation, migration, and phenotypic switching in VSMCs. Mechanistically, STING-NF-κB signaling was involved in this process. The pharmacological inhibition of STING induced by C-176 partially prevented neointima formation due to suppression of VSMCs proliferation. Taken together, STING-NF-κB axis significantly promoted proliferation, migration, and phenotypic switching of VSMCs, which may be a novel therapeutic approach to combat vascular proliferative diseases.

Galangin inhibits neointima formation induced by vascular injury via regulating the PI3K/AKT/mTOR pathway.

Aims: The proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in the pathological process of neointima formation after vascular injury. Galangin, an extract of the ginger plant galangal, is involved in numerous biological activities, including inhibiting the proliferation and migration of tumor cells, but its effect on VSMCs is unknown. This study focused on the role and mechanism of galangin in the neointima formation induced by vascular injury. Methods and results: In this study, we found that galangin restrained the PDGF-BB-induced proliferation, migration and phenotypic switching of VSMCs in a concentration-dependent manner. In vivo, we established a model of carotid artery balloon injury in rats, followed by intragastric administration of galangin (40 mg kg-1 day-1 or 80 mg kg-1 day-1) for 14 or 28 consecutive days. Then, the degree of neointima hyperplasia was evaluated by H&E staining, and the level of relevant protein expression was assessed by immunofluorescence and western blotting. In vitro, we isolated and grew primary rat aortic smooth muscle cells, which were treated with PDGF-BB and different doses of galangin, and then CCK-8 assay, wound healing assay, transwell assay, western blotting and immunofluorescence assays were performed. We found that galangin significantly inhibited PDGF-BB-induced proliferation, migration, and phenotypic switching of VSMCs and promoted autophagy in VSMCs in vitro, and galangin significantly inhibited neointimal hyperplasia after the common carotid artery balloon injury in rats. In terms of mechanisms, galangin inhibited the PI3K/AKT/mTOR pathway, thereby suppressing VSMC's switch from a contractile to a synthetic phenotype, inhibiting VSMC proliferation, migration and phenotypic switching and upregulating the Beclin1 protein expression levels and the ratio of LC3BII/I, promoting VSMC autophagy, and thereby inhibiting neointimal hyperplasia after vascular injury. Conclusion: Our study suggests that galangin inhibits neointimal hyperplasia after vascular injury by inhibiting smooth muscle cell proliferation, migration and phenotypic switching and by promoting autophagy, and that galangin may be a promising drug for the prevention and treatment of vascular restenosis after PCI.

DN200434 Inhibits Vascular Smooth Muscle Cell Proliferation and Prevents Neointima Formation in Mice after Carotid Artery Ligation.

BACKGRUOUND: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which contributes to the development of occlusive vascular diseases, requires elevated mitochondrial oxidative phosphorylation to meet the increased requirements for energy and anabolic precursors. Therefore, therapeutic strategies based on blockade of mitochondrial oxidative phosphorylation are considered promising for treatment of occlusive vascular diseases. Here, we investigated whether DN200434, an orally available estrogen receptor-related gamma inverse agonist, inhibits proliferation and migration of VSMCs and neointima formation by suppressing mitochondrial oxidative phosphorylation. METHODS: VSMCs were isolated from the thoracic aortas of 4-week-old Sprague-Dawley rats. Oxidative phosphorylation and the cell cycle were analyzed in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using a Seahorse XF-24 analyzer and flow cytometry, respectively. A model of neointimal hyperplasia was generated by ligating the left common carotid artery in male C57BL/6J mice. RESULTS: DN200434 inhibited mitochondrial respiration and mammalian target of rapamycin complex 1 activity and consequently suppressed FBS- or PDGF-stimulated proliferation and migration of VSMCs and cell cycle progression. Furthermore, DN200434 reduced carotid artery ligation-induced neointima formation in mice. CONCLUSION: Our data suggest that DN200434 is a therapeutic option to prevent the progression of atherosclerosis.

Systemic single administration of anti-inflammatory microRNA 146a-5p loaded in polymeric nanomedicines with active targetability attenuates neointimal hyperplasia by controlling inflammation in injured arteries in a rat model.

Neointimal hyperplasia (NIH) after revascularization is a key unsolved clinical problem. Various studies have shown that attenuation of the acute inflammatory response on the vascular wall can prevent NIH. MicroRNA146a-5p (miR146a-5p) has been reported to show anti-inflammatory effects by inhibiting the NF-κB pathway, a well-known key player of inflammation of the vascular wall. Here, a nanomedicine, which can reach the vascular injury site, based on polymeric micelles was applied to deliver miR146a-5p in a rat carotid artery balloon injury model. In vitro studies using inflammation-induced vascular smooth muscle cell (VSMC) was performed. Results showed anti-inflammatory response as an inhibitor of the NF-κB pathway and VSMC migration, suppression of reactive oxygen species production, and proinflammatory cytokine gene expression in VSMCs. A single systemic administration of miR146a-5p attenuated NIH and vessel remodeling in a carotid artery balloon injury model in both male and female rats in vivo. MiR146a-5p reduced proinflammatory cytokine gene expression in injured arteries and monocyte/macrophage infiltration into the vascular wall. Therefore, miR146a-5p delivery to the injury site demonstrated therapeutic potential against NIH after revascularization.

α-Iso-cubebene attenuates neointima formation by inhibiting HMGB1-induced monocyte to macrophage differentiation via suppressing ROS production.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis, a medicinal herb used to improve cardiovascular symptoms. To investigate the mechanisms involved, the effects of ICB on cellular production of reactive oxygen species (ROS) was determined using cultured human THP-1 cells. When THP-1 cells were stimulated with HMGB1, cellular concentration of ROS was increased in dose- and time-dependent manners. These increases were significantly attenuated in cells pretreated with NADPH oxidase inhibitors, diphenyleneiodonium chloride and apocynin, but not by other inhibitors related to ROS generation in monocytes. The expression of constitutively expressed NADPH oxidase (NOX) subunits including NOX1, NOX2, NOX4 and NOX5 was not affected by HMGB1, but HMGB1-induced ROS production was exclusively attenuated in NOX2-deficient cells using siRNA, suggesting an enhanced NOX2 complex assembly. When cells were stimulated with HMGB1, p47phox phosphorylation at ser345, ser359 and ser370 was increased in dose- and time-dependent manners, which were significantly attenuated in ICB (3-10 µg/mL)-pretreated cells. In addition, HMGB1-induced monocyte-macrophage differentiation (MMD) in bone marrow-derived cells isolated from mice were significantly attenuated in cells treated with apocynin and ICB. Also, macrophage infiltration and intimal hyperplasia in the wire-injured femoral artery were significantly attenuated in ICB-treated mice compared to wild-type control mice. The results of this study show that ICB inhibits HMGB1-induced MMD by suppressing ROS production in monocytes, thus suggest that ICB has therapeutic potential for vascular inflammation with subsequent intimal hyperplasia related to vascular injury.