Inhibits Neointima Formation Research Articles

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.

Detoxification and Activating Blood Circulation Decoction Promotes Reendothelialization of Damaged Blood Vessels via VEGF Signaling Pathway Activation by miRNA-126.

The occurrence of in-stent restenosis (ISR) poses a significant challenge for percutaneous coronary intervention (PCI). Thus, the promotion of vascular reendothelialization is essential to inhibit endothelial proliferation. In this study, we clarified the mechanism by which Detoxification and Activating Blood Circulation Decoction (DABCD) promotes vascular reendothelialization to avoid ISR by miRNA-126-mediated modulation of the vascular endothelial growth factor (VEGF) signaling pathway. A rat model of post-PCI restenosis was established by balloon injury. The injured aortic segment was collected 14 and 28 d after model establishment. Our findings indicate that on the 14th and 28th days following balloon injury, DABCD reduced intimal hyperplasia and inflammation and promoted vascular reendothelialization. Additionally, DABCD markedly increased nitric oxide (NO) expression and significantly decreased ET-1 production in rat serum. DABCD also increased the mRNA level of endothelial nitric oxide synthase (eNOS) and the protein expression of VEGF, p-Akt, and p-extracellular signal-regulated kinase (ERK)1/2 in vascular tissue. Unexpectedly, the expression of miR-126a-5p mRNA was significantly lower in the aortic tissue of balloon-injured rats than in the aortic tissue of control rats, and higher miR-126a-5p levels were observed in the DABCD groups. The results of this study indicated that the vascular reendothelialization effect of DABCD on arterial intimal injury is associated with the inhibition of neointimal formation and the enhancement of vascular endothelial activity. More specifically, the effects of DABCD were mediated, at least in part, through miR-126-mediated VEGF signaling pathway activation.

Abstract 650: Disrupting Neointimal Formation In Mouse And Human Vascular Tissues Through Targeting Connexin 43

Vascular damage caused by stent placement triggers smooth muscle cell (SMC) de-differentiation, leading to an aggressively proliferating phenotype that thickens the vessel wall and blocks the artery, which contributes to stent failure. We have identified the gap junction protein connexin 43 (Cx43) as a significant contributor to neointima progression. Phosphorylation of Cx43 by mitogen-activated protein kinase (MAPK) promotes its binding to the cell cycle regulator cyclin E and its kinase CDK2, promoting SMC proliferation. Beyond this, we do not know how these proteins regulate SMC proliferation in humans. We tested the hypothesis that disrupting Cx43-cyclin E binding could reduce neointima formation in human blood vessels. We used peptide arrays to identify cyclin E binding sites on Cx43 and to define critical Cx43 MAPK-phosphorylation residues that control their interactions. Based on this, we created a 15 amino acid, stearate-linked, cell-permeable, connexin mimetic peptide termed CycliCx that binds cyclin E. Experiments were controlled using Cx43 mimetic peptides designed against regions not bound by cyclin E. In cultured human coronary artery SMC, the CycliCx peptide reduces Cx43 MAPK-phosphorylation, disrupts Cx43-cyclin E interactions, and prevents PDGF-induced SMC proliferation. Application of the CycliCx peptide to mouse carotid arteries inhibits neointima formation in a 14-day ligation-induced neointima model (n=8, male and female). We developed an ex vivo model of PDGF-induced neointimal formation in excess human saphenous vein tissues recovered after CABG surgery. In the human saphenous vein preparations treated with PDGF, the CycliCx peptide disrupts neointima formation after 14 days (n=9). In conclusion, our data suggest that disrupting Cx43-cyclin E interactions provides protection against pathological SMC proliferation. These data highlight Cx43 as a potential drug target to prevent human neointima development.

Paeonol Promotes Reendothelialization After Vascular Injury Through Activation of c-Myc/VEGFR2 Signaling Pathway.

Dysfunction of endothelium is associated with multiple pathological vascular diseases. However, how to regulate reendothelialization after vascular injury is not well defined. This study aims to determine whether and how Paeonol controls reendothelialization following artery injury. The endothelium of murine carotid artery was denuded by catheter guide wires injury. H&E staining and IF staining were performed to determine whether Paeonol is critical for reendothelialization. BRDU Incorporation Assay, Boyden Chamber Migration Assay, Tube Formation Assay, and Spheroid Sprouting Assay were used to investigate whether Paeonol is involved in regulating proliferation and migration of endothelial cells. The underlying mechanism of how Paeonol regulates reendothelialization was determined by Molecular docking simulation and CO-IP Assay. Paeonol treatment significantly inhibits neointima formation in carotid artery ligation model by promoting proliferation and migration of endothelial cells. Mechanistically, Paeonol enhances c-Myc expression, consequently interacts with VEGFR2 results in activating VEGF signaling pathway, and eventually promotes reendothelialization after vascular injury. Our data demonstrated that Paeonol plays a critical role in regulating vascular reendothelialization, which may be therapeutically used for treatment of pathological vascular diseases.

Polo-like kinase 4 inhibitor CFI-400945 inhibits carotid arterial neointima formation but increases atherosclerosis

Neointima lesion and atherosclerosis are proliferative vascular diseases associated with deregulated proliferation of vascular smooth muscle cells (SMCs). CFI-400945 is a novel, highly effective anticancer drug that inhibits polo-like kinase 4 (PLK4) and targets mitosis. In this study, we aim to investigate how CFI-400945 affects the development of proliferative vascular diseases. In C57BL/6 mice, neointima formation was generated by complete carotid ligation. In apolipoprotein E knockout (ApoE−/−) mice fed a high-fat diet, atherosclerosis was induced by partial carotid ligation. CFI-400945 was directly applied to carotid arteries via a perivascular collar. Our results showed that CFI-400945 drastically inhibited neointima formation but significantly accelerated atherosclerosis. In vitro studies showed that CFI-400945 treatment induced SMC polyploidization and arrested cells in the G2/M phase. CFI-400945 treatment upregulated p53 and p27 expression but decreased p21 and cyclin B1 expression. CFI-400945 also induced SMC apoptosis, which was inhibited by hydroxyurea, a DNA synthesis inhibitor that inhibits polyploidization. Furthermore, CFI-400945 caused supernumerary centrosomes, leading to mitotic failure, resulting in polyploidization. In conclusion, CFI-400945 prevents carotid arterial neointima formation in C57BL/6 mice but accelerates atherosclerosis in ApoE−/− mice, likely through mitotic arrest and subsequent induction of polyploidization and apoptosis.

MKL1 fuels ROS-induced proliferation of vascular smooth muscle cells by modulating FOXM1 transcription

Reactive oxygen species (ROS) promotes vascular injury and neointima formation in part by stimulating proliferation of vascular smooth muscle cells (VSMC). The underlying transcriptional mechanism, however, is not completely understood. Here we report that VSMC-specific deletion of MKL1 in mice suppressed neointima formation in a classic model of vascular injury. Likewise, pharmaceutical inhibition of MKL1 activity by CCG-1423 similarly mollified neointima formation in mice. Over-expression of a constitutively active MKL1 in vascular smooth muscle cells enhanced proliferation in a ROS-dependent manner. On the contrary, MKL1 depletion or inhibition attenuated VSMC proliferation. PCR array based screening identified forkhead box protein M1 (FOXM1) as a direct target for MKL1. MKL1 interacted with E2F1 to activate FOXM1 expression. Concordantly, FOXM1 depletion ameliorated MKL1-dependent VSMC proliferation. Of interest, ROS-induced MKL1 phosphorylation through MK2 was essential for its interaction with E2F1 and consequently FOXM1 trans-activation. Importantly, a positive correlation between FOXM1 expression and VSMC proliferation was identified in arterial specimens from patients with restenosis. Taken together, our data suggest that a redox-sensitive phosphorylation-switch of MKL1 activates FOXM1 transcription and mediates ROS fueled vascular smooth muscle proliferation. Targeting the MK-2/MKL1/FOXM1 axis may be considered as a reasonable approach for treatment of restenosis.

Long Noncoding RNA TPRG1-AS1 Suppresses Migration of Vascular Smooth Muscle Cells and Attenuates Atherogenesis via Interacting With MYH9 Protein.

Migration of human aortic smooth muscle cells (HASMCs) contributes to the pathogenesis of atherosclerosis. This study aims to functionally characterize long noncoding RNA TPRG1-AS1 (tumor protein p63 regulated 1, antisense 1) in HASMCs and reveal the underlying mechanism of TPRG1-AS1 in HASMCs migration, neointima formation, and subsequent atherosclerosis. The expression of TPRG1-AS1 in atherosclerotic plaques was verified a series of in silico analysis and quantitative real-time polymerase chain reaction analysis. Northern blot, rapid amplification of cDNA ends and Sanger sequencing were used to determine its full length. In vitro transcription-translation assay was used to investigate the protein-coding capacity of TPRG1-AS1. RNA fluorescent in situ hybridization was used to confirm its subcellular localization. Loss- and gain-of-function studies were used to investigate the function of TPRG1-AS1. Furthermore, the effect of TPRG1-AS1 on the pathological response was evaluated in carotid balloon injury model, wire injury model, and atherosclerosis model, respectively. TPRG1-AS1 was significantly increased in atherosclerotic plaques. TPRG1-AS1 did not encode any proteins and its full length was 1279nt, which was bona fide a long noncoding RNA. TPRG1-AS1 was mainly localized in cytoplasmic and perinuclear regions in HASMCs. TPRG1-AS1 directly interacted with MYH9 (myosin heavy chain 9) protein in HASMCs, promoted MYH9 protein degradation through the proteasome pathway, hindered F-actin stress fiber formation, and finally inhibited HASMCs migration. Vascular smooth muscle cell-specific transgenic overexpression of TPRG1-AS1 significantly reduced neointima formation, and attenuated atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. This study demonstrated that TPRG1-AS1 inhibited HASMCs migration through interacting with MYH9 protein and consequently suppressed neointima formation and atherosclerosis.

Systemic Delivery of Clopidogrel Inhibits Neointimal Formation in a Mouse Vein Graft Model.

Clopidogrel inhibits platelet aggregation and has beneficial effects on patients undergoing coronary artery bypass grafting surgery, but it is unknown whether clopidogrel inhibits the neointima formation of grafted veins. In this study, we used a murine vein graft model to study the effect of clopidogrel on intima hyperplasia of the vein graft. Vein grafting was performed among C57BL/6J mice, immediately after surgery; 1 mg/kg clopidogrel and vehicle control were used to inject mice peritoneally daily for 2 weeks. As compared with the vehicle, clopidogrel significantly inhibited the neointima formation of vein grafts at 4 weeks after surgeries. The immunohistochemistry study showed that as compared with the vehicle, clopidogrel significantly decreased the rate of proliferating cell nuclear antigen-positive cells in the wall of vein grafts and significantly increased the expression of vascular smooth muscle cell (VSMC) contractile protein markers (α-smooth muscle actin, calponin, and SM22) within the neointima area of vein grafts. Clopidogrel significantly decreased the plasma interleukin 6 (IL-6) level at 1 week after surgery as compared with the vehicle. We isolated VSMCs from mouse aortic arteries. As compared with the vehicle, clopidogrel significantly inhibited thrombin-induced VSMC proliferation and migration, significantly decreased IL-6 mRNA expression and protein secretion, and increased intracellular cyclic adenosine monophosphate generation in a dose-dependent manner. In conclusion, systemic delivery of clopidogrel inhibits neointima formation of the mouse vein graft, the mechanisms of which are associated with its inhibitory effects on VSMC proliferation, migration, and the tendency to synthetic phenotype after vein graft surgery, reducing the expression of IL-6 and increasing the intracellular cyclic adenosine monophosphate level.

Theaflavin-3,3'-Digallate from Black Tea Inhibits Neointima Formation Through Suppression of the PDGFRβ Pathway in Vascular Smooth Muscle Cells.

The abnormal neointima formation caused by the phenotypic switching of vascular smooth cells (VSMCs) into a synthetic state plays a key role in the pathogenesis of various vascular diseases, including atherosclerosis and postangioplasty restenosis. Theaflavin-3,3′-digallate (TF3) in black tea has been reported to exert antiinflammatory and anticancer effects, but its role in neointima formation remains unclear. Here, we delineated a remarkable effect of TF3 in suppressing neointima formation of VSMCs in vivo as well as the ability of primary rat aortic smooth cells (RASMCs) to proliferate and migrate in vitro. Further study confirmed that the effects of TF3 on PDGF-BB–induced RASMCs were due to reduced phosphorylation of PDGFRβ, which led to the repression of downstream pathways. We concluded that TF3 may act as a repressor in the progression of neointima formation and serve as a potential therapeutic candidate for excessive phenotypic switching of VSMCs.

The lncRNA Punisher Regulates Apoptosis and Mitochondrial Homeostasis of Vascular Smooth Muscle Cells via Targeting miR-664a-5p and OPA1.

Long noncoding RNAs (lncRNAs) are important regulators of various cellular functions. Recent studies have shown that a novel lncRNA termed Punisher is highly expressed in cardiovascular progenitors and has potential role in cardiovascular diseases. However, its role, especially in molecular mechanism, is unclear. In our present study, we observed that Punisher was obviously downregulated in atherosclerotic plaques. Further research proved that it can suppress the apoptosis of VSMCs potentially contributing to the progression of atherosclerosis. Intriguingly, Punisher revealed to regulate mitochondria fission as well as mitochondrial functions induced by hydrogen peroxide (H2O2) in VSMCs. Mechanistically, Punisher was further proved to serve as a ceRNA which directly binds to miR-664a-5p and consequently regulates its target OPA1, and finally contributes to the biological function of VSMCs. Particularly, Punisher overexpression distinctly suppressed neointima formation and VSMC apoptosis in vivo. Encouragingly, these results were in accordance with findings obtained with the clinical evaluation of patients with atherosclerosis. Our data provides the significant relationship among OPA1, mitochondrial homeostasis, VSMC apoptosis, and atherosclerosis. And lncRNA Punisher and miR-664a-5p could serve as the novel and potential targets in the diagnosis and treatment of cardiovascular diseases.

Randomized Trial of Chocolate Touch Compared With Lutonix Drug-Coated Balloon in Femoropopliteal Lesions (Chocolate Touch Study).

First-generation drug-coated balloons (DCBs) have significantly reduced the rate of restenosis compared with balloon angioplasty alone; however, high rates of bailout stenting and dissections persist. The Chocolate Touch DCB is a nitinol constrained balloon designed to reduce acute vessel trauma and inhibit neointima formation and restenosis. Patients with claudication or ischemic rest pain (Rutherford class 2-4) and superficial femoral or popliteal disease (≥70% stenosis) were randomized 1:1 to Chocolate Touch or Lutonix DCB at 34 sites in the United States, Europe, and New Zealand. The primary efficacy end point was DCB success, defined as primary patency at 12 months (peak systolic velocity ratio <2.4 by duplex ultrasound without clinically driven target lesion revascularization in the absence of clinically driven bailout stenting). The primary safety end point was freedom from major adverse events at 12 months, a composite of target limb-related death, major amputation, or reintervention. Both primary end points were tested for noninferiority, and if met, sequential superiority testing for efficacy followed by safety was prespecified. An independent clinical events committee, and angiographic and duplex ultrasound core laboratories blinded to treatment allocation reviewed all end points. A total of 313 patients were randomized to Chocolate Touch (n=152) versus Lutonix DCB (n=161). Follow-up at 1 year was available in 94% of patients. The mean age was 69.4±9.5 years, the average lesion length was 78.1±46.9 mm, and 46.2% had moderate-to-severe calcification. The primary efficacy rates of DCB success at 12 months was 78.8% (108/137) with Chocolate Touch and 67.7% (88/130) with Lutonix DCB (difference, 11.1% [95% CI, 0.6-21.7]), meeting noninferiority (Pnoninferiority<0.0001) and sequential superiority (Psuperiority=0.04). The primary safety event rate was 88.9% (128/144) with Chocolate Touch and 84.6% (126/149) with Lutonix DCB (Pnoninferiority<0.001; Psuperiority=0.27). In this prospective, multicenter, randomized trial, the second-generation Chocolate Touch DCB met both noninferiority end points for efficacy and safety and was more effective than Lutonix DCB at 12 months for the treatment of femoropopliteal disease. URL: https://www. gov; Unique identifier: NCT02924857.

MEK1/2 inhibitor inhibits neointima formation by activating miR-126-3p/ C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor 4 (CXCR4) axis

ABSTRACT Endothelial dysfunction is an initial and essential step in vascular-remodeling diseases, including atherosclerosis and neointima formation. During vascular remodeling, activated endothelial cells can release pro-inflammatory factors that promote phenotypic switching of vascular smooth muscle cells (VSMCs) to the proliferative phenotype. We previously reported that MEK1/2 inhibitor, U0126, has a protective effect on the development of atherosclerosis and vascular calcification. However, the effect of MEK1/2 inhibitors on neointimal formation and the underlying mechanism is not fully understood. We determined that MEK1/2 inhibitor reduced carotid artery ligation-induced neointimal formation, while increased collagen and elastin levels and vascular integrality. Mechanistically, MEK1/2 inhibitor or ERK1/2 siRNA increased miR-126-3p level in endothelial cells, thereby inhibiting expression of regular of G-protein signaling 16 (RGS16), a miR-126-3p target gene, to activate the C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor 4 (CXCR4) signaling pathway. Accordingly, miR-126-3p was also increased by U0126 in serum and carotid artery. RGS16 was inhibited while CXCR4 and CXCL12 was increased by U0126 in neointimal areas, especially in the endothelium. Moreover, similar results were observed in atherosclerotic plaques of high-fat diet-fed apolipoprotein E deficiency (apoE−/−) mice. In addition, vascular cell adhesion molecule 1 (VCAM-1), another miR-126-3p target gene, was reduced by U0126 in the neointimal areas, resulting reduced monocytes/macrophages accumulation. Taken together, our results indicate that MEK1/2 inhibitor can reduce neointima formation by activating endothelial miR-126-3p production to facilitate endothelium repair while reduce monocyte adhesion/infiltration.

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.

PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1−/− VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1−/− VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.

Abstract MP28: The Bet Bromodomain Inhibitor, (+)-JQ1, Inhibits Neointima Formation Following Acute Vascular Injury Via Pten Upregulation

The primary aim of this work was to identify compounds that upregulate phosphatase and tensin homolog (PTEN) expression in smooth muscle cells (SMCs), with the expectation that PTEN upregulation would inhibit pathological vascular remodeling by promoting SMC homeostasis. Our recent work demonstrated that PTEN is an important regulator of SMC phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Angiotensin-II-induced pathological vascular remodeling, suggesting that pharmacologic PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Currently, there are few known pharmacologic inducers of PTEN, however, we have shown that (+)-JQ1, which targets the epigenetic reader protein, Brd4, directly activates the PTEN promoter to drive PTEN expression. Mechanistically, our findings suggest that Brd4 inhibition results in p300-mediated hyper-acetylation of the PTEN promoter to increase PTEN transcriptional activity. Administration of (+)-JQ1 or SMC-specific Brd4 deletion inhibited neointima formation following acute carotid artery injury in mice. Using SMC-specific PTEN depletion both in vitro and in vivo, we showed that the observed anti-remodeling, anti-inflammatory effects were PTEN-dependent. Furthermore, we tested the effects of (+)-JQ1 on macrophage polarization, as systemic drug delivery could potentially modulate the phenotype of other resident vascular cells, including macrophages. We found that (+)-JQ1 inhibited bone marrow-derived macrophage polarization towards a pro-inflammatory phenotype, which is notable because vascular inflammation driven by SMC-macrophage cross-talk is known to promote neointima development. Importantly, our findings may hold direct clinical relevance, as BET bromodomain inhibitors related to (+)-JQ1 are currently undergoing clinical trials. These results are significant because they indicate that targeting PTEN upregulation in SMCs is a completely novel approach to inhibit vascular remodeling.

Deletion of Ulk1 inhibits neointima formation by enhancing KAT2A/GCN5-mediated acetylation of TUBA/α-tubulin in vivo

ABSTRACT ULK1 (unc-51 like autophagy activating kinase) has a central role in initiating macroautophagy/autophagy, a process that contributes to atherosclerosis and neointima hyperplasia, or excessive tissue growth that leads to vessel dysfunction. However, the role of ULK1 in neointima formation remains unclear. We aimed to determine how Ulk1 deletion affected neointima formation and to investigate the underlying mechanisms. We measured autophagy activity, vascular smooth muscle cell (VSMC) migration and neointima hyperplasia in cultured VSMCs and ligation-injured mouse carotid arteries from male wild-type (WT, C57BL/6 J) and VSMC-specific ulk1 knockout (ulk1 KO) mice. Carotid artery ligation in WT mice increased ULK1 protein expression, and concurrently increased autophagic flux and neointima formation. Treating human aortic smooth muscle cells (HASMCs) with PDGF (platelet derived growth factor) increased ULK1 expression, activated autophagy, and promoted cell migration. Further, smooth muscle cell-specific deletion of Ulk1 suppressed autophagy, inhibited VSMC migration, and impeded neointima hyperplasia. Mechanistically, Ulk1 deletion inhibited autophagic degradation of histone acetyltransferase protein KAT2A/GCN5 (K[lysine] acetyltransferase 2A), resulting in accumulation of KAT2A that directly acetylated TUBA/α-tubulin and subsequently increased protein levels of acetylated TUBA. The acetylation of TUBA increased microtubule stability and inhibited VSMC directional migration and neointima formation. Finally, local transfection of Kat2a siRNA decreased TUBA acetylation and prevented the attenuation of vascular injury-induced neointima formation in ulk1 KO mice. These findings suggest that Ulk1 deletion inhibits neointima formation by reducing autophagic degradation of KAT2A and increasing TUBA acetylation in VSMCs. Abbreviations: ACTA2/α-SMA: actin, alpha 2, smooth muscle, aorta; ACTB: actin beta; ATAT1: alpha tubulin acetyltransferase 1; ATG: autophagy related; BECN1: beclin 1; BP: blood pressure; CAL: carotid artery ligation; CQ: chloroquine diphosphate; EC: endothelial cells; EEL: external elastic layer; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HASMCs: human aortic smooth muscle cells; HAT1: histone acetyltransferase 1; HDAC: histone deacetylase; IEL: inner elastic layer; IP: immunoprecipitation; KAT2A/GCN5: K(lysine) acetyltransferase 2A; KAT8/hMOF: lysine acetyltransferase 8; MAP1LC3: microtubule associated protein 1 light chain 3; MYH11: myosin heavy chain 11; PBS: phosphate-buffered saline; PDGF: platelet derived growth factor; PECAM1/CD31: platelet and endothelial cell adhesion molecule 1; RAC3: Rac family small GTPase 3; SIRT2: sirtuin 2; SPP1/OPN: secreted phosphoprotein 1; SQSTM1/p62: sequestosome 1; TAGLN/SM22: transgelin; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase; VSMC: vascular smooth muscle cell; VVG: Verhoeff Van Gieson; WT: wild type.

Roles of vascular endothelial and smooth muscle cells in the vasculoprotective effect of insulin in a mouse model of restenosis.

Background:Insulin exerts vasculoprotective effects on endothelial cells (ECs) and growth-promoting effects on vascular smooth muscle cells (SMCs) in vitro, and suppresses neointimal growth in vivo. Here we determined the role of ECs and SMCs in the effect of insulin on neointimal growth.Methods:Mice with transgene CreERT2 under the control of EC-specific Tie2 (Tie2-Cre) or SMC-specific smooth muscle myosin heavy chain promoter/enhancer (SMMHC-Cre) or littermate controls were crossbred with mice carrying a loxP-flanked insulin receptor (IR) gene. After CreERT2-loxP-mediated recombination was induced by tamoxifen injection, mice received insulin pellet or sham (control) implantation, and underwent femoral artery wire injury. Femoral arteries were collected for morphological analysis 28 days after wire injury.Results:Tamoxifen-treated Tie2-Cre+ mice showed lower IR expression in ECs, but not in SMCs, than Tie2-Cre− mice. Insulin treatment reduced neointimal area after arterial injury in Tie2-Cre− mice, but had no effect in Tie2-Cre+ mice. Tamoxifen-treated SMMHC-Cre+ mice showed lower IR expression in SMCs, but not in ECs, than SMMHC-Cre− mice. Insulin treatment reduced neointimal area in SMMHC-Cre− mice, whereas unexpectedly, it failed to inhibit neointima formation in SMMHC-Cre+ mice.Conclusion:Insulin action in both ECs and SMCs is required for the “anti-restenotic” effect of insulin in vivo.

Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

[Figure: see text].

Knockout of the NONO Gene Inhibits Neointima Formation in a Mouse Model of Vascular Injury.

[Figure: see text].

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury

In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.