Healing the injured vessel wall using microRNA-facilitated gene delivery

Drug-eluting stents have emerged as potent weapons in the treatment of patients with symptomatic coronary artery disease by reducing restenosis rates; however, a significant clinical consequence of these stents is delayed reendothelialization, which may increase the risk of late stent thrombosis. In this issue of the JCI, Santulli and colleagues generated an adenovirus that expresses the cyclin-dependent kinase inhibitor p27^Kip1 (p27) and bears four tandem copies of target sequences for the endothelial cell–enriched microRNA (miRNA) miR-126-3p (Ad-p27-126TS) in an attempt to specifically reduce proliferation of vascular smooth muscle cells, but not endothelial cells. Indeed, delivery of Ad-p27-126TS to balloon-injured arteries in rats not only induced faster and more complete reendothelialization, but also effectively improved neointimal hyperplasia, hypercoagulability, and vasoreactivity. Collectively, these findings provide a cogent foundation for the potential therapeutic use of miRNA-facilitated gene delivery strategies to heal vessel wall injury.     

Repression of Choroidal Neovascularization Through Actin Cytoskeleton Pathways by MicroRNA-24

Actin cytoskeleton is critical for cell motility and division, both of which are important for angiogenesis. MicroRNAs (miRNA/miR) are emerging as pivotal modulators of vascular development and disease. How miRNAs regulate actin cytoskeleton dynamics in endothelial cells (EC) and neovascularization is still unclear. Here, we report that miR-24 regulates actin dynamics in ECs through targeting multiple members downstream of Rho signaling, including Pak4, Limk2, and Diaph1 proteins. Overexpression of miR-24 in ECs blocks stress fiber and lamellipodia formation, represses EC migration, proliferation, and tube formation in vitro, as well as angiogenesis in an ex vivo aortic ring assay. Moreover, subretinal delivery of miR-24 mimics represses laser-induced choroidal neovascularization (CNV) in vivo. Mechanistically, knockdown of miR-24 target protein LIMK2 or PAK4 inhibits stress fiber formation and tube formation in vitro, mimicking miR-24 overexpression phenotype in angiogenesis, while overexpression of LIMK2 and PAK4 by adenoviruses partially rescued the tube formation defects in miR-24 overexpressing ECs. Taken together, these findings suggest that miR-24 represses angiogenesis by simultaneously regulating multiple components in the actin cytoskeleton pathways. Manipulation of actin cytoskeleton pathways by miR-24 may represent an attractive therapeutic solution for the treatment of wet age-related macular degeneration (AMD) and other vascular diseases.     

SM22α启动子靶向性调控血管平滑肌细胞过表达p27的研究

目的:构建由SM22α特异性启动子介导血管平滑肌细胞(VSMCs)靶向性过表达p27蛋白的慢病毒载体来研究对VSMCs细胞周期的影响。方法:构建重组慢病毒载体(Lenti-SM22α-p27-EGFP),感染原代培养的VSMCs及内皮细胞(VECs)。结果:SM22α-p27慢病毒能对VSMCs表现出高效且精确的抑制作用。SM22α启动子靶向标记的p27能在VSMCs内过表达,且通过G0/G1期阻滞来抑制细胞增殖。结论:由SM22α特异性启动子驱动的重组慢病毒载体能选择性感染VSMCs,有效地引起p27蛋白过表达并产生G0/G1期阻滞。     

MicroRNA-181b regulates NF-κB-mediated vascular inflammation

EC activation and dysfunction have been linked to a variety of vascular inflammatory disease states. The function of microRNAs (miRNAs) in vascular EC activation and inflammation remains poorly understood. Herein, we report that microRNA-181b (miR-181b) serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium by targeting importin-α3, a protein that is required for nuclear translocation of NF-κB. Overexpression of miR-181b inhibited importin-α3 expression and an enriched set of NF-κB-responsive genes such as adhesion molecules VCAM-1 and E-selectin in ECs in vitro and in vivo. In addition, treatment of mice with proinflammatory stimuli reduced miR-181b expression. Rescue of miR-181b levels by systemic administration of miR-181b "mimics" reduced downstream NF-κB signaling and leukocyte influx in the vascular endothelium and decreased lung injury and mortality in endotoxemic mice. In contrast, miR-181b inhibition exacerbated endotoxin-induced NF-κB activity, leukocyte influx, and lung injury. Finally, we observed that critically ill patients with sepsis had reduced levels of miR-181b compared with control intensive care unit (ICU) subjects. Collectively, these findings demonstrate that miR-181b regulates NF-κB-mediated EC activation and vascular inflammation in response to proinflammatory stimuli and that rescue of miR-181b expression could provide a new target for antiinflammatory therapy and critical illness.     

Transmembrane protein ESDN promotes endothelial VEGF signaling and regulates angiogenesis

Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell–derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VE-cadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.     

lncRNA RPL34-AS1调控miR-670-5p/SMAD4对胃癌细胞生物学行为的影响

目的探讨lncRNA RPL34-AS1对胃癌细胞生物学行为的影响及其对miR-670-5p/SMAD4分子轴的调控作用。方法回顾性收集2018年5月至2019年6月期间临沂市肿瘤医院收治的33例胃癌患者的癌组织及其癌旁组织标本。QRT-PCR检测RPL34-AS1、miR-670-5p的表达量,蛋白质印迹法检测SMAD4表达。体外培养人胃癌细胞HGC-27,实验分组:pc DNA组(转染RPL34-AS1过表达载体的阴性对照)、pc DNA-RPL34-AS1组(转染RPL34-AS1过表达载体)、anti-miR-NC组(转染miR-670-5p特异性寡核苷酸抑制剂的阴性对照)、anti-miR-670-5p组(转染miR-670-5p特异性寡核苷酸抑制剂)、pc DNA-RPL34-AS1+miR-NC组(共转染pc DNA-RPL34-AS1与阴性对照mimic NC序列)、pc DNA-RPL34-AS1+miR-670-5p组(共转染pc DNA-RPL34-AS1与miR-670-5p寡核苷酸模拟物)。MTT与平板克隆形成实验检测细胞增殖与克隆形成数,流式细胞术检测细胞凋...     

Circular RNA PPP1CC promotes Porphyromonas gingivalis-lipopolysaccharide-induced pyroptosis of vascular smooth muscle cells by activating the HMGB1/TLR9/AIM2 pathway

ObjectivePorphyromonas gingivalis (Pg) plays a critical role in the occurrence and development of atherosclerosis. Lipopolysaccharide from Pg (Pg-LPS) could lead to pyroptosis of vascular smooth muscle cells (VSMCs) and induce instability of atherosclerotic plaque. Therefore, pyroptosis of VSMCs could promote the process of atherosclerosis. However, the exact mechanism of Pg-LPS-induced pyroptosis of VSMCs is unclear.MethodsWe determined pyroptosis and expression of interleukin (IL)-1β and IL-18 in VSMCs using 4′,6-diamidino-2-phenylindole staining and ELISA after stimulation by Pg-LPS. We established a knockdown plasmid containing the circular (circ)RNA PPP1CC and transfected it into VSMCs. Luciferase assays were performed to reveal the association between microRNAs miR-103a-3p and miR-107 and circRNA PPP1CC.ResultsStimulation of Pg-LPS led to pyroptosis of VSMCs. Knockdown of circRNA PPP1CC relieved the Pg-LPS-induced pyroptosis of VSMCs and suppressed the expression of HMGB1, TLR9, AIM2, and cleaved caspase-1. Luciferase assays showed that PPP1CC directly targeted and competitively adsorbed miR-103a-3p and miR-107, weakening the inhibitory effect of these microRNAs on the expression of HMGB1.ConclusionKnockdown of circRNA PPP1CC relieved Pg-LPS-induced pyroptosis of VSMCs. Pyroptosis of VSMCs appears to promote atherosclerosis and may represent a novel therapeutic target for its treatment.     

Fibroblast growth factors: the keepers of endothelial normalcy

Endothelial cells (ECs) under physiologic and pathologic conditions are capable of substantial plasticity that includes the endothelial-mesenchymal transition (EndMT). Notably, in the hypoxic pulmonary circulation EndMT likely drives increases in the pulmonary arterial blood pressure, leading to pulmonary arterial hypertension (PAH). However, it is unclear whether suppressing EndMT can prevent PAH development or mitigate established disease. In this issue of the JCI, Woo et al. generated mice with EC-specific deletion of FGFR1 and -2 and mice with EC-specific expression of a constitutively active FGFR1 to determine the role of FGF signaling in PAH. Mice with FGFR1/2 deletion in ECs that were exposed to hypoxic conditions developed extensive EndMT and more severe PAH than control mice. Animals with the constitutively active endothelial FGFR were protected from hypoxia-induced EndMT and PAH development. These findings suggest that FGF signaling may promote vascular resilience and prevent hypoxia-induced development of EndMT and PAH.     

Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides.

Restenosis after angioplasty is due predominantly to accumulation of vascular smooth muscle cells (VSMCs). The resistance of restenosis to pharmacological treatment has prompted investigation of genes involved in VSMC proliferation. We have examined the effect on VSMC proliferation of blocking expression of the c-myc proto-oncogene with antisense oligodeoxynucleotides, both in vitro and in a rat carotid artery injury model of angioplasty restenosis. Antisense c-myc oligodeoxynucleotides reduced average cell levels of c-myc mRNA and protein by 50-55% and inhibited proliferation of VSMCs when mitogenically stimulated from quiescence or when proliferating logarithmically (IC50 = 10 micrograms/ml). Corresponding sense c-myc, two-base-pair mismatch antisense c-myc, antisense alpha-actin or glyceraldehyde phosphate dehydrogenase oligodeoxynucleotides did not suppress c-myc expression or inhibit VSMC proliferation. Antisense c-myc inhibition was relieved by overexpression of an exogenous c-myc gene. After balloon catheter injury, peak c-myc mRNA expression occurred at 2 h. Antisense c-myc applied in a pluronic gel to the arterial adventitia reduced peak c-myc expression by 75% and significantly reduced neointimal formation at 14 d, compared with sense c-myc and gel application alone. We conclude that c-myc expression is required for VSMC proliferation in vitro and in the vessel wall. C-myc is a therefore a potential target for adjunctive therapy to reduce angioplasty restenosis.     

LncRNA NEXN-AS1 attenuates proliferation and migration of vascular smooth muscle cells through sponging miR-33a/b

Non-protein-coding RNAs (lncRNAs) are emerging as important regulators in disease pathogenesis, including atherosclerosis (AS). Here, we investigated the role and underlying mechanisms of nexilin F-actin binding protein antisense RNA 1 (NEXN-AS1) on the proliferation and migration of vascular smooth muscle cells (VSMCs). Our data revealed that ox-LDL treatment resulted in decreased NEXN-AS1 expression and increased miR-33a/b levels in human aorta VSMCs (HA-VSMCs) in dose- and time-dependent manners. Overexpression of NEXN-AS1 mitigated the proliferation and migration of HA-VSMCs under ox-LDL stimulation using CCK-8 and wound-healing assays. Moreover, dual-luciferase reporter and RNA immunoprecipitation assays verified that NEXN-AS1 acted as molecular sponges of miR-33a and miR-33b in HA-VSMCs. MiR-33a or miR-33b silencing attenuated the proliferation and migration of ox-LDL-treated HA-VSMCs. Furthermore, miR-33a or miR-33b mediated the inhibitory effects of NEXN-AS1 overexpression on the proliferation and migration of ox-LDL-treated HA-VSMCs. Our study suggested that high level of NEXN-AS1 mitigated VSMC proliferation and migration under ox-LDL stimulation at least partly through sponging miR-33a and miR-33b, illuminating NEXN-AS1 as a novel therapeutic approach for AS treatment.

Non-protein-coding RNAs (lncRNAs) are emerging as important regulators in disease pathogenesis, including atherosclerosis (AS).     

Epothilone B inhibits neointimal formation after rat carotid injury through the regulation of cell cycle-related proteins

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury model and examined the potential effects of epothilone B on cardiovascular disease. We found that epothilone B potently prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly inhibited 5% fetal bovine serum (FBS)- and 50 ng/ml platelet-derived growth factor (PDGF)-BB-induced proliferation and cell cycle progression in rat aortic VSMCs. Furthermore, FBS and PDGF-BB induced the activations of extracellular signal-regulated kinases 1 and 2, Akt, phospholipase C gamma 1, and PDGF-receptor beta chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant decrease in the level of cyclin-dependent protein kinase (CDK) 2, whereas it caused no change in the levels of cyclin E and down-regulated the phosphorylation of retinoblastoma, which plays a critical role in cell cycle regulation. Furthermore, levels of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of VSMCs. These findings suggest that epothilone B can inhibit neointimal formation via the cell cycle arrest by the regulation of the cell cycle-related proteins in VSMCs.     

腺病毒介导hRAMP1基因转染血管平滑肌细胞的增殖和凋亡

背景：外源性受体活性修饰蛋白1基因转染可能对血管平滑肌细胞增殖调控具有重要的作用。目的：探讨腺病毒载体介导人受体活性修饰蛋白1基因对体外培养的兔血管平滑肌细胞增殖和凋亡的影响。方法：分离培养获得兔主动脉血管平滑肌细胞,分别以携带人受体活性修饰蛋白1基因的腺病毒和空腺病毒转染后,分成人受体活性修饰蛋白1组、空病毒组和对照组。结果与结论：腺病毒转染细胞后随时间延长,报告基因绿色荧光蛋白表达逐渐增加,72h表达最强,感染率达80%,96h时仍有绿色荧光蛋白表达。人受体活性修饰蛋白1组的受体活性修饰蛋白1蛋白表达增加,血管平滑肌细胞凋亡率增加,细胞增殖明显抑制,与空病毒组和对照组比较差异有显著性意义（P〈0.05）。提示人受体活性修饰蛋白1基因感染血管平滑肌细胞后明显抑制血管平滑肌细胞增殖,促进细胞凋亡。     

MiR-96 promotes proliferation and chemo- or radioresistance by down-regulating RECK in esophageal cancer

The involvement of miR-96 in esophageal cancer (EC) remains unclear. The aim of this study is to explore the functional role of miR-96 and determine whether miR-96 could be a potential therapeutic target for human esophageal cancer. MiR-96 up-regulation was demonstrated in 145 EC samples and RECK down-regulation was validated in EC cell lines. Moreover, ectopic overexpression of miR-96 in TE-1 or ECa-109 contributed to tumor growth in xenograft mouse models. Furthermore, up-regulation of miR-96 could reduce the susceptibilities of EC cells to chemotherapy or radiotherapy. RECK was identified as a target of miR-96 and RECK overexpressing could abrogate the growth of EC cells induced by miR-96. Taken together, miR-96 serves as an oncogene role in EC cells through downregulating RECK.     

Overexpression of PDGF-BB decreases colorectal and pancreatic cancer growth by increasing tumor pericyte content

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.     

miR-146b-5p对骨肉瘤细胞增殖抑制及克隆形成的机制研究

目的　检测骨肉瘤组织中 miR-146b-5p表达，探究其对骨肉瘤（ osteosarcoma，OS）细胞增殖、克隆形成的影响及潜在分子作用机制。方法　选取 2018年 1月～ 2020年 12月内蒙古自治区巴彦淖尔市医院病理科 30组临床骨肉瘤组织及邻近正常组织标本，采用实时荧光定量 PCR实验（ qRT-PCR）法检测组织中 miR-146b-5p表达水平；通过介导转染 miR-146b-5p mimics和 miR-146b-5p ASO分别过表达和敲低 miR-146b-5p表达，探究其对骨肉瘤细胞增殖及克隆形成的影响。通过生物学信息数据库预测 miR-146b-5p的潜在靶基因，荧光素酶实验验证两者结合关系，分析 miR-146b5p对靶基因的调控作用。分析靶基因在骨肉瘤中的表达特征及与 miR-146b-5p的相关性，通过细胞增殖实验验证两者的调控作用，以其探究在骨肉瘤中发挥功能的潜在分子机制。结果　骨肉瘤组织中 miR-146b-5p表达（ 4.096±1.237）显著低于邻近正常组织（ 5.895±0.834），差异有统计学意义（ t=6.605，P＜ 0.001）。miR-146b-5p过表达抑制了骨肉瘤细胞的增殖能力（ t=13.233～ 34.599，均 P＜ 0.01）。miR-146b-5p过表达组克隆形成数目（ 48.912±2.032）较对照组（160.834±9.031）明显降低，差异有统计学意义（ t=20.942，P＜ 0.001）。DUSP16是 miR-146b-5p的潜在靶基因， miR-146b-5p负向调控双特异性磷酸酶（ dual speci.city phosphatase 16, DUSP16）表达。骨肉瘤组织中 DUSP16表达（5.683±0.457）较邻近正常组织（ 4.665±0.531）显著升高，差异有统计学意义（ t=7.959，P＜ 0.001），与 miR-146b5p表达呈负相关（ r=-0.667，P<0.05）。共转染过表达 DUSP16逆转了 miR-146b-5p对骨肉瘤细胞增殖的抑制作用。 miR-146b-5p过表达组 P21水平（ 4.995±0.214）较对照组（ 1.001±0.002）显著升高，差异有统计学意义（ t=32.325，P＜ 0.001）；当共转染 DUSP16过表达后， P21表达（ 0.986±0.012）较单独过表达 miR-146b-5p组（4.995±0.214）显著降低，差异有统计学意义（ t=32.398，P＜ 0.001）。结论　骨肉瘤组织中 miR-146b-5p显著低表达，其过表达抑制了骨肉瘤细胞的增殖及克隆形成； miR-146b-5p通过靶向负调控 DUSP16表达参与 P53信号通路从而在骨肉瘤发生发展起重要作用。     

KIS protects against adverse vascular remodeling by opposing stathmin-mediated VSMC migration in mice

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27^Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27^Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS^–/– mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27^Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS^–/– VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.