lncRNA H19 facilitates vascular neointima formation by targeting miR-125a-3p/FLT1 axis.

The aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of neointima formation in vascular restenosis. This study aims to explore the function of the long noncoding RNA H19 in neointima formation. A mouse carotid ligation model was established, and human vascular smooth muscle cells (VSMCs) were used as a cell model. lncRNA H19 overexpression promoted VSMC proliferation and migration. Moreover, miR-125a-3p potentially bound to lncRNA H19, and Fms-like tyrosine kinase-1 (FLT1) might be a direct target of miR-125a-3p in VSMCs. Upregulation of miR-125a-3p alleviated lncRNA H19-enhanced VSMC proliferation and migration. Furthermore, rescue experiments showed that enhanced expression of miR-125a-3p attenuated lncRNA H19-induced FLT1 expression in VSMCs. In addition, the overexpression of lncRNA H19 significantly exacerbated neointima formation in a mouse carotid ligation model. In summary, lncRNA H19 stimulates VSMC proliferation and migration by acting as a competing endogenous RNA (ceRNA) of miR-125a-3p. lncRNA H19 may be a therapeutic target for restenosis.

Spatiotemporal EP4-fibulin-1 expression is associated with vascular intimal hyperplasia.

AIMS: Cyclooxygenase-2-derived prostaglandin E2 (PGE2) is thought to promote vascular intimal hyperplasia (IH). It has been reported that the PGE2 receptor EP4 is upregulated in injured vessels, and that EP4 signaling in vascular smooth muscle cells (VSMCs) promotes IH. In contrast, EP4 in endothelial cells has been demonstrated to restrain IH. We aimed to investigate spatiotemporal expression of EP4 and whether modulating EP4 signaling could be a viable therapeutic strategy. METHODS AND RESULTS: We generated EP4 reporter mice (Ptger4-IRES-nlsLacZ) and found temporary but prominent EP4 expression in VSMCs of the proliferative neointima 2 weeks after femoral artery wire injury. Injury-induced IH was diminished in VSMC-targeted EP4 heterozygous deficient mice (Ptger4fl/+; SM22-Cre) 2 and 4 weeks after vascular injury compared to that in SM22-Cre, whereas injury-induced IH was exacerbated in VSMC-targeted EP4-overexpressing mice (Ptger4-Tg) compared to controls (non-Tg). We then investigated the downstream signaling of EP4 in VSMCs. Stimulation of EP4 increased mRNA and protein levels of the glycoprotein fibulin-1 in Ptger4-Tg VSMCs. Fibulin-1C recombinant proteins increased VSMC proliferation and migration through transforming growth factor (TGF)-ß/Smad3, and EP4-mediated proliferation and migration were attenuated in Fbln1fl/fl; SM22-Cre VSMCs and in CRISPR/Cas9-mediated Fbln1 knockdown in Ptger4-Tg VSMCs. We generated multiple deletion mutants of fibulin-1C and found that EGF-like modules 6-8 appear to be involved in fibulin-1-mediated proliferation. Among binding partners of fibulin-1, extracellular matrix protein 1 (ECM1) was also upregulated by EP4 stimulation, and fibulin-1C and ECM1 proteins additively enhanced VSMC proliferation and migration. Injury-induced IH was attenuated in VSMC-targeted fibulin-1 deletion mice (Fbln1fl/fl; SM22-Cre) compared to Fbln1fl/fl. Furthermore, systemic EP4 antagonist administration reduced injury-induced IH in wild-type mice. CONCLUSIONS: EP4 was upregulated in VSMCs of proliferative IH, and EP4 signaling promoted IH, at least in part through fibulin-1. An EP4 antagonist might be considered as a therapeutic strategy for IH.

NONRATT000538.2 promotes vascular smooth muscle cell phenotypic switch and in-stent restenosis.

Vascular smooth muscle cell (VSMC) excessive proliferation and migration are considered the main pathological process in in-stent restenosis (ISR) following vascular intervention. Certain long noncoding RNAs play vital roles in this process. Therefore, this study aimed to explore novel regulators for ISR and further uncover the mechanism. Using a rat abdominal aorta stent implantation model, we observed that NONRATT000538.2 (NR538.2) served as a positive regulator for VSMC proliferation and migration. By manipulating NR538.2 expression via adenoviral overexpression or siRNA knockdown, we noted that NR538.2 promoted VSMC phenotypic switching, thereby inducing proliferation and migration. Significantly, the local delivery of siRNA of NR538.2 via adeno-associated virus vector suppressed balloon injury-induced neointima formation. Our study demonstrated for the first time that NR538.2 positively influenced VSMC proliferation during ISR.

Early thrombus formation is required for eccentric and heterogeneous neointimal hyperplasia under disturbed flow.

BACKGROUND: Anticoagulation and antiplatelet therapy effectively inhibit neointimal hyperplasia (NIH) in both arterial and venous systems but not in arteriovenous fistulae (AVF). The main site of AVF failure is the juxta-anastomotic area that is characterized by disturbed flow compared with laminar flow in the arterial inflow and the venous outflow. OBJECTIVES: We hypothesized that early thrombus formation is required for eccentric and heterogeneous NIH in the presence of disturbed flow. METHODS: Needle puncture and sutured AVF were created in C57BL/6 mice, in PF4-Cre × mT/mG reporter mice, and in Wistar rats. Human AVF samples were second-stage basilic vein transpositions. The tissues were examined by histology, immunofluorescence, immunohistochemistry, and en face staining. RESULTS: In the presence of disturbed flow, both mouse and human AVF showed eccentric and heterogeneous NIH. Maladapted vein wall was characterized by eccentric and heterogeneous neointima that was composed of a different abundance of thrombus and smooth muscle cells. PF4-cre × mT/mG reporter mice AVF showed that green fluorescent protein-labeled platelets deposit on the wall directly facing the fistula exit with endothelial cell loss and continue to accumulate in the presence of disturbed flow. Neither disturbed flow with limited endothelial cell loss nor nondisturbed flow induced heterogeneous neointima in different animal models. CONCLUSION: Early thrombus contributes to late heterogeneous NIH in the presence of disturbed flow. Disturbed flow, large area of endothelial cell loss, and thrombus formation are critical to form eccentric and heterogeneous NIH. Categorization of adapted or maladapted walls may be helpful for therapy targeting heterogeneous NIH.

KIF11 promotes vascular smooth muscle cell proliferation by regulating cell cycle progression and accelerates neointimal formation after arterial injury in mice.

Background and aims: One of the primary causes of lumen narrowing is vascular injury induced during medical procedures. Vascular injury disrupts the integrity of the endothelium, triggering platelet deposition, leukocyte recruitment, and the release of inflammatory factors. This, in turn, induces the proliferation of vascular smooth muscle cells (VSMCs), leading to neointima formation. However, the molecular mechanism underlying VSMC proliferation following injury remains unknown. KIF11 is critical in regulating the cell cycle by forming bipolar spindles during mitotic metaphase. This process may contribute to VSMCs proliferation and neointima formation following vascular injury. Yet, the function of KIF11 in VSMCs has not been elucidated. This study aims to investigate the role and mechanisms of KIF11 in regulating VSMCs cycle progression and proliferation. Methods: After conducting biological analysis of the transcriptome sequencing data from the mouse carotid artery injury model and the cell transcriptome data of PDGF-BB-induced VSMCs, we identified a potential target gene, KIF11, which may play a crucial role in vascular injury. Then we established a vascular injury model to investigate how changes in KIF11 expression and activity influence in vivo VSMCs proliferation and neointimal formation. In addition, we employed siRNA and specific inhibitors to suppress KIF11 expression and activity in VSMCs cultured in vitro to study the mechanisms underlying VSMCs cycle progression and proliferation. Results: The results of immunohistochemistry and immunofluorescence indicate a significant upregulation of KIF11 expression in the injured vascular. The intraperitoneal injection of the KIF11 specific inhibitor, K858, partially inhibits intimal hyperplasia in the vascular injury model. In vitro experiments further demonstrate that PDGF-BB upregulates KIF11 expression through the PI3K/AKT pathway, and enhances KIF11 activity. Inhibition of both KIF11 expression and activity partially reverses the pro-cycle progression and pro-proliferation effects of PDGF-BB on VSMCs. Additionally, KIF11 overexpression partially counteracts the proliferation arrest and cell cycle arrest induced by inhibiting the PI3K/AKT pathway in VSMCs. Conclusion: Our study highlights the crucial role of KIF11 in regulating the cycle progression and proliferation of VSMCs after vascular injury. A comprehensive understanding of these mechanisms could pave the way for potential therapeutic interventions in treating vascular stenosis.

MIRO1 controls energy production and proliferation of smooth muscle cells.

Background: The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. Methods: An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). Results: MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Conclusions: Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

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.

Dynamic changes in proresolving lipid mediators and their receptors following acute vascular injury in male rats.

Acute vascular injury provokes an inflammatory response, resulting in neointimal hyperplasia (NIH) and downstream pathologies. The resolution of inflammation is an active process in which specialized proresolving lipid mediators (SPM) and their receptors play a central role. We sought to examine the acute phase response of SPM and their receptors in both circulating blood and the arterial wall in a rat angioplasty model. We found that the ratio of proresolving to pro-inflammatory lipid mediators (LM) in plasma decreased sharply 1 day after vascular injury, then increased slightly by day 7, while that in arteries remained depressed. Granulocyte expression of SPM receptors ALX/FPR2 and DRV2/GPR18, and a leukotriene B4 receptor BLT1 increased postinjury, while ERV1/ChemR23 expression was reduced early and then recovered by day 7. Importantly, we show unique arterial expression patterns of SPM receptors in the acute setting, with generally low levels through day 7 that contrasted sharply with that of the pro-inflammatory CCR2 receptor. Overall, these data document acute, time-dependent changes of LM biosynthesis and SPM receptor expression in plasma, leukocytes, and artery walls following acute vascular injury. A biochemical imbalance between inflammation and resolution LM pathways appears persistent 7 days after angioplasty in this model. These findings may help guide therapeutic approaches to accelerate vascular healing and improve the outcomes of vascular interventions for patients with advanced atherosclerosis.

Inhibition of endothelial to mesenchymal transition in a large animal preclinical arterio-venous fistula model leads to improved remodeling and reduced stenosis.

AIMS: Vein grafts are used for many indications, including bypass graft surgery and arterio-venous fistula (AVF) formation. However, patency following vein grafting or AVF formation is suboptimal for various reasons, including thrombosis, neointimal hyperplasia and adverse remodeling. Recently, endothelial to mesenchymal transition (EndMT) was found to contribute to neointimal hyperplasia in mouse vein grafts. We aimed to evaluate the clinical potential of inhibiting EndMT, and developed the first dedicated preclinical model to study the efficacy of local EndMT inhibition immediately prior to AVF creation. METHODS AND RESULTS: We first undertook pilot studies to optimize the creation of a femoral AVF in pigs and verify that EndMT contributes to neointimal formation. We then developed a method to achieve local in vivo SMAD3 knockdown by dwelling a lentiviral construct containing SMAD3 shRNA in the femoral vein prior to AVF creation. Next, in Phase 1, 6 pigs were randomized to SMAD3 knockdown or control lentivirus to evaluate the effectiveness of SMAD3 knockdown and EndMT inhibition 8 days after AVF creation. In Phase 2, 16 pigs were randomized to SMAD3 knockdown or control lentivirus and were evaluated to assess longer-term effects on AVF diameter, patency and related measures at 30 days after AVF creation.In Phase 1, compared to controls, SMAD3 knockdown achieved a 75% reduction in the proportion of CD31+ endothelial cells co-expressing SMAD3 (p<0.001), and also a significant reduction in the extent of EndMT (p<0.05). In Phase 2, compared to controls, SMAD3 knockdown was associated with an increase in the minimum diameter of the venous limb of the AVF (1.56±1.66 versus 4.26±1.71mm, p<0.01) and a reduced degree of stenosis (p<0.01). Consistent with this, neointimal thickness was reduced in the SMAD3 knockdown group (0.88±0.51 versus 0.45±0.19mm, p<0.05). Furthermore, endothelial integrity (the proportion of luminal cells expressing endothelial markers) was improved in the SMAD3 knockdown group (p<0.05). CONCLUSIONS: EndMT inhibition in a preclinical AVF model by local SMAD3 knockdown using gene therapy led to reduced neointimal hyperplasia, increased endothelialization and a reduction in the degree of AVF stenosis. This provides important proof-of-concept to pursue this approach as a clinical strategy to improve the patency of AVFs and other vein grafts.

Inositol 1,4,5-Trisphosphate Receptors Regulate Vascular Smooth Muscle Cell Proliferation and Neointima Formation in Mice.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is involved in many types of arterial diseases, including neointima hyperplasia, in which Ca2+ has been recognized as a key player. However, the physiological role of Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs) from endoplasmic reticulum in regulating VSMC proliferation has not been well determined. METHODS AND RESULTS: Both in vitro cell culture models and in vivo mouse models were generated to investigate the role of IP3Rs in regulating VSMC proliferation. Expression of all 3 IP3R subtypes was increased in cultured VSMCs upon platelet-derived growth factor-BB and FBS stimulation as well as in the left carotid artery undergoing intimal thickening after vascular occlusion. Genetic ablation of all 3 IP3R subtypes abolished endoplasmic reticulum Ca2+ release in cultured VSMCs, significantly reduced cell proliferation induced by platelet-derived growth factor-BB and FBS stimulation, and also decreased cell migration of VSMCs. Furthermore, smooth muscle-specific deletion of all IP3R subtypes in adult mice dramatically attenuated neointima formation induced by left carotid artery ligation, accompanied by significant decreases in cell proliferation and matrix metalloproteinase-9 expression in injured vessels. Mechanistically, IP3R-mediated Ca2+ release may activate cAMP response element-binding protein, a key player in controlling VSMC proliferation, via Ca2+/calmodulin-dependent protein kinase II and Akt. Loss of IP3Rs suppressed cAMP response element-binding protein phosphorylation at Ser133 in both cultured VSMCs and injured vessels, whereas application of Ca2+ permeable ionophore, ionomycin, can reverse cAMP response element-binding protein phosphorylation in IP3R triple knockout VSMCs. CONCLUSIONS: Our results demonstrated an essential role of IP3R-mediated Ca2+ release from endoplasmic reticulum in regulating cAMP response element-binding protein activation, VSMC proliferation, and neointima formation in mouse arteries.

Inhibition of Vascular Smooth Muscle Cell Proliferation by ENPP1: The Role of CD73 and the Adenosine Signaling Axis.

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.

Potential of dapagliflozin to prevent vascular remodeling in the rat carotid artery following balloon injury.

BACKGROUND AND AIMS: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular events independently of glycemic control. However, the possibility that SGLT2 inhibitors improve vascular restenosis is unknown. The aim of this study was to examine whether dapagliflozin could prevent neointima thickening following balloon injury and, if so, to determine the underlying mechanisms. METHODS: Saline, dapagliflozin (1.5 mg/kg/day), or losartan (30 mg/kg/day) was administered orally for five weeks to male Wistar rats. Balloon injury of the left carotid artery was performed a week after starting the treatment and rats were sacrificed 4 weeks later. The extent of neointima was assessed by histomorphometric and immunofluorescence staining analyses. Vascular reactivity was assessed on injured and non-injured carotid artery rings, changes of target factors by immunofluorescence, RT-qPCR, and histochemistry. RESULTS: Dapagliflozin and losartan treatments reduced neointima thickening by 32 % and 27 %, respectively. Blunted contractile responses to phenylephrine and relaxations to acetylcholine and down-regulation of eNOS were observed in the injured arteries. RT-qPCR investigations indicated an increased in gene expression of inflammatory (IL-1beta, VCAM-1), oxidative (p47phox, p22phox) and fibrotic (TGF-beta1) markers in the injured carotid. While these changes were not affected by dapagliflozin, increased levels of AT1R and NTPDase1 (CD39) and decreased levels of ENPP1 were observed in the restenotic carotid artery of the dapagliflozin group. CONCLUSIONS: Dapagliflozin effectively reduced neointimal thickening. The present data suggest that dapagliflozin prevents restenosis through interfering with angiotensin and/or extracellular nucleotides signaling. SGLT2 represents potential new target for limiting vascular restenosis.

Loss of Smooth Muscle Tenascin-X Inhibits Vascular Remodeling Through Increased TGF-ß Signaling.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.

AKAP1 alleviates VSMC phenotypic modulation and neointima formation by inhibiting Drp1-dependent mitochondrial fission.

The roles and mechanisms of A-kinase anchoring protein 1 (AKAP1) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. AKAP1 is a mitochondrial PKA-anchored protein and maintains mitochondrial homeostasis. This study aimed to investigate how AKAP1/PKA signaling plays a protective role in inhibiting VSMC phenotypic transformation and neointima formation by regulating mitochondrial fission. The results showed that both PDGF-BB treatment and balloon injury reduced the transcription, expression, and mitochondrial anchoring of AKAP1. In vitro, the overexpression of AKAP1 significantly inhibited PDGF-BB mediated VSMC proliferation and migration, whereas AKAP1 knockdown further aggravated VSMC phenotypic transformation. Additionally, in the balloon injury model in vivo, AKAP1 overexpression reduced neointima formation, the muscle fiber area ratio, and rat VSMC proliferation and migration. Furthermore, PDGF-BB and balloon injury inhibited Drp1 phosphorylation at Ser637 and promoted Drp1 activity and mitochondrial midzone fission; AKAP1 overexpression reversed these effects. AKAP1 overexpression also inhibited the distribution of mitochondria at the plasma membrane and the reduction of PKARIIß expression induced by PDGF-BB, as evidenced by an increase in mitochondria-plasma membrane distance as well as PKARIIß protein levels. Moreover, the PKA agonist promoted Drp1 phosphorylation (Ser637) and inhibited PDGF-BB-mediated mitochondrial fission, cell proliferation, and migration. The PKA antagonist reversed the increase in Drp1 phosphorylation (Ser637) and the decline in mitochondrial midzone fission and VSMC phenotypic transformation caused by AKAP1 overexpression. The results of this study reveal that AKAP1 protects VSMCs against phenotypic modulation by improving Drp1 phosphorylation at Ser637 through PKA and inhibiting mitochondrial fission, thereby preventing neointima formation.

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.

The white-collar sign after Neuroform Atlas stent-assisted coil embolization of unruptured intracranial aneurysms.

PURPOSE: Although stent-assisted technique is expected to help provide a scaffold for neointima formation at the orifice of the aneurysm, not all aneurysms treated with stent-assisted technique develop complete neointima formation. The white-collar sign (WCS) indicates neointimal tissue formation at the aneurysm neck that prevents aneurysm recanalization. The aim of this study was to explore factors related to WCS appearance after stent-assisted coil embolization of unruptured intracranial aneurysms (UIAs). METHODS: A total of 59 UIAs treated with a Neuroform Atlas stent were retrospectively analyzed. The WCS was identified on digital subtraction angiography (DSA) 1 year after coil embolization. The cohort was divided into WCS-positive and WCS-negative groups, and possible predictors of the WCS were explored using logistic regression analysis. RESULTS: The WCS appeared in 20 aneurysms (33.9%). In the WCS-positive group, neck size was significantly smaller (4.2 (interquartile range (IQR): 3.8-4.6) versus 5.4 (IQR: 4.2-6.8) mm, p = .006), the VER was significantly higher (31.8% (IQR: 28.6%-38.4%) versus 27.6% (IQR: 23.6%-33.8%), p = .02), and the rate of RROC class 1 immediately after treatment was significantly higher (70% vs 20.5%, p < .001) than in the WCS-negative group. On multivariate analysis, neck size (odds ratio (OR): 0.542, 95% confidence interval (CI): 0.308-0.954; p = .03) and RROC class 1 immediately after treatment (OR: 6.99, 95% CI: 1.769-27.55; p = .006) were independent predictors of WCS appearance. CONCLUSIONS: Smaller neck size and complete occlusion immediately after treatment were significant factors related to WCS appearance in stent-assisted coil embolization for UIAs using the Neuroform Atlas stent.

Transcription Factor 21: A Transcription Factor That Plays an Important Role in Cardiovascular Disease.

BACKGROUND: According to the World Health Organisation's Health Report 2019, approximately 17.18 million people die from cardiovascular disease each year, accounting for more than 30% of all global deaths. Therefore, the occurrence of cardiovascular disease is still a global concern. The transcription factor 21 (TCF21) plays an important role in cardiovascular diseases. This article reviews the regulation mechanism of TCF21 expression and activity and focuses on its important role in atherosclerosis in order to contribute to the development of diagnosis and treatment of cardiovascular diseases. SUMMARY: TCF21 is involved in the phenotypic regulation of vascular smooth muscle cells (VSMCs), promotes the proliferation and migration of VSMCs, and participates in the activation of inflammatory sequences. Increased proliferation and migration of VSMCs can lead to neointimal hyperplasia after vascular injury. Abnormal hyperplasia of neointima and inflammation are one of the main features of atherosclerosis. Therefore, targeting TCF21 may become a potential treatment for relieving atherosclerosis. KEY MESSAGES: TCF21 as a member of basic helix-loop-helix transcription factors regulates cell growth and differentiation by modulating gene expression during the development of different organs and plays an important role in cardiovascular development and disease. VSMCs and cells derived from VSMCs constitute the majority of plaques in atherosclerosis. TCF21 plays a key role in regulation of VSMCs' phenotype, thus accelerating atherogenesis in the early stage. However, TCF21 enhances plaque stability in late-stage atherosclerosis. The dual role of TCF21 should be considered in the translational medicine.

Small nucleolar RNA host gene 18 controls vascular smooth muscle cell contractile phenotype and neointimal hyperplasia.

AIMS: Long non-coding RNA (LncRNA) small nucleolar RNA host gene 18 (SNHG18) has been widely implicated in cancers. However, little is known about its functional involvement in vascular diseases. Herein, we attempted to explore a role for SNHG18 in modulating vascular smooth muscle cell (VSMC) contractile phenotype and injury-induced neointima formation. METHODS AND RESULTS: Analysis of single-cell RNA sequencing and transcriptomic datasets showed decreased levels of SNHG18 in injured and atherosclerotic murine and human arteries, which is positively associated with VSMC contractile genes. SNHG18 was upregulated in VSMCs by TGFß1 through transcription factors Sp1 and SMAD3. SNHG18 gene gain/loss-of-function studies revealed that VSMC contractile phenotype was positively regulated by SNHG18. Mechanistic studies showed that SNHG18 promotes a contractile VSMC phenotype by up-regulating miR-22-3p. SNHG18 up-regulates miR-22 biogenesis and miR-22-3p production by competitive binding with the A-to-I RNA editing enzyme, adenosine deaminase acting on RNA-2 (ADAR2). Surprisingly, we observed that ADAR2 inhibited miR-22 biogenesis not through increasing A-to-I editing within primary miR-22, but by interfering with the binding of microprocessor complex subunit DGCR8 to primary miR-22. Importantly, perivascular SNHG18 overexpression in the injured vessels dramatically up-regulated the expression levels of miR-22-3p and VSMC contractile genes, and prevented injury-induced neointimal hyperplasia. Such modulatory effects were reverted by miR-22-3p inhibition in the injured arteries. Finally, we observed a similar regulator role for SNHG18 in human VSMCs and a decreased expression level of both SNHG18 and miR-22-3p in diseased human arteries; and we found that the expression level of SNHG18 was positively associated with that of miR-22-3p in both healthy and diseased human arteries. CONCLUSION: We demonstrate that SNHG18 is a novel regulator in governing VSMC contractile phenotype and preventing injury-induced neointimal hyperplasia. Our findings have important implications for therapeutic targeting snhg18/miR-22-3p signalling in vascular diseases.

Gallic Acid Inhibits Proliferation and Migration of Smooth Muscle Cells in a Pig In-Stent Restenosis Model.

In-stent restenosis (ISR) develops primarily due to neointimal hyperplasia. Gallic acid (GA) has anti-inflammatory, antioxidant, and cardioprotective effects. This study sought to investigate the effects of GA on neointimal hyperplasia and proliferation and migration of vascular smooth muscle cells (VSMCs) in a pig ISR model. In vitro proliferation and migration experiments were confirmed, after VSMCs were treated with platelet-derived growth factor (PDGF-BB) and GA (100 µM) using a 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay and a scratch wound assay for 24 hours and 48 hours. A bare metal stent (BMS) was implanted in the pig coronary artery to induce ISR with overdilation (1.1-1.2:1), and GA (10 mg/kg/day) was administered for 4 weeks. At the 4-week follow-up, optical coherence tomography (OCT) and histopathological analyses were performed. GA decreased the proliferation of VSMCs by PDGF-BB for 24 hours (89.24±24.56% vs. 170.04±19.98%, p<0.001) and 48 hours (124.87±7.35% vs. 187.64±4.83%, p<0.001). GA inhibited the migration of VSMCs induced by PDGF-BB for 24 hours (26.73±2.38% vs. 65.38±9.73%, p<0.001) and 48 hours (32.96±3.04% vs. 77.04±10.07%, p<0.001). Using OCT, % neointimal hyperplasia was shown to have significantly decreased in the GA group compared with control vehicle group (28.25±10.07% vs. 37.60±10.84%, p<0.001). GA effectively reduced neointimal hyperplasia by inhibiting the proliferation and migration of VSMCs in a pig ISR model. GA could be a potential treatment strategy for reducing ISR after stent implantation.