Vascular Proliferative Diseases Research Articles

H3K4 Methyltransferase Smyd3 Mediates Vascular Smooth Muscle Cell Proliferation, Migration, and Neointima Formation.

[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.

Translational Research in Retinopathy of Prematurity: From Bedside to Bench and Back Again.

Retinopathy of prematurity (ROP), a vascular proliferative disease affecting preterm infants, is a leading cause of childhood blindness. Various studies have investigated the pathogenesis of ROP. Clinical experience indicates that oxygen levels are strongly correlated with ROP development, which led to the development of oxygen-induced retinopathy (OIR) as an animal model of ROP. OIR has been used extensively to investigate the molecular mechanisms underlying ROP and to evaluate the efficacy of new drug candidates. Large clinical trials have demonstrated the efficacy of anti-vascular endothelial growth factor (VEGF) agents to treat ROP, and anti-VEGF therapy is presently becoming the first-line treatment worldwide. Anti-VEGF therapy has advantages over conventional treatments, including being minimally invasive with a low risk of refractive error. However, long-term safety concerns and the risk of late recurrence limit this treatment. There is an unmet medical need for novel ROP therapies, which need to be addressed by safe and minimally invasive therapies. The recent progress in biotechnology has contributed greatly to translational research. In this review, we outline how basic ROP research has evolved with clinical experience and the subsequent emergence of new drugs. We discuss previous and ongoing trials and present the candidate molecules expected to become novel targets.

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling-Brief Report.

The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-β, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

CircSFMBT2 facilitates vascular smooth muscle cell proliferation by targeting miR-331-3p/HDAC5

ObjectiveTo investigate the functional role of circSFMBT2 in vascular smooth muscle cell (VSMC) proliferation and migration and the underlying molecular mechanism. MethodsThe circSFMBT2 levels in neointimal tissue and platelet derived growth factor-BB (PDGF-BB)-treated VSMCs were detected by qRT-PCR. The role of circSFMBT2 in VSMC proliferation, migration and cell cycle distribution was assessed by MTT assay, transwell assay, wound healing assay and flow cytometry. The protein expression of contractile markers was evaluated by western blot. In vitro luciferase reporter assay, RNA pull-down assay, ChIP and coimmunoprecipitation (CoIP) were performed to explore the effects of circSFMBT2 on the downstream signaling pathway. ResultsWe found that circSFMBT2 was markedly increased in neointimal tissue relative to normal tissue and PDGF-BB-treated VSMCs relative to control VSMCs. The knockdown of circSFMBT2 by siRNA significantly inhibited the proliferation and migration of VSMCs. Interestingly, circSFMBT2 knockdown enhanced the expression of contractile marker proteins including SM22α, SM myosin heavy chain (SMMHC) and calponin. Further data demonstrated that circSFMBT2 interacted with miR-331-3p as a competing endogenous RNA and up-regulated the expression of histone deacetylase 5 (HDAC5), thereby regulating the level of angiogenic factor with G patch and FHA domains (Aggf1). ConclusionThese results revealed that circSFMBT2 plays a vital role in VSMC proliferation and migration through the miR-331/HDAC5/Aggf1 axis, and suggest a novel target for treating proliferative vascular diseases.

The SGLT2 inhibitor Empagliflozin attenuates interleukin-17A-induced human aortic smooth muscle cell proliferation and migration by targeting TRAF3IP2/ROS/NLRP3/Caspase-1-dependent IL-1β and IL-18 secretion

Chronic inflammation and persistent oxidative stress contribute to the development and progression of vascular proliferative diseases. We hypothesized that the proinflammatory cytokine interleukin (IL)-17A induces oxidative stress and amplifies inflammatory signaling in human aortic smooth muscle cells (SMC) via TRAF3IP2-mediated NLRP3/caspase-1-dependent mitogenic and migratory proinflammatory cytokines IL-1β and IL-18. Further, we hypothesized that these maladaptive changes are prevented by empagliflozin (EMPA), an SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor. Supporting our hypotheses, exposure of cultured SMC to IL-17A promoted proliferation and migration via TRAF3IP2, TRAF3IP2-dependent superoxide and hydrogen peroxide production, NLRP3 expression, caspase-1 activation, and IL-1β and IL-18 secretion. Furthermore, NLRP3 knockdown, caspase-1 inhibition, and pretreatment with IL-1β and IL-18 neutralizing antibodies and IL-18BP, each attenuated IL-17A-induced SMC migration and proliferation. Importantly, SMC express SGLT2, and pre-treatment with EMPA attenuated IL-17A/TRAF3IP2-dependent oxidative stress, NLRP3 expression, caspase-1 activation, IL-1β and IL-18 secretion, and SMC proliferation and migration. Importantly, silencing SGLT2 attenuated EMPA-mediated inhibition of IL-17A-induced cytokine secretion and SMC proliferation and migration. EMPA exerted these beneficial antioxidant, anti-inflammatory, anti-mitogenic and anti-migratory effects under normal glucose conditions and without inducing cell death. These results suggest the therapeutic potential of EMPA in vascular proliferative diseases.

Carnosine Impedes PDGF-Stimulated Proliferation and Migration of Vascular Smooth Muscle Cells In Vitro and Sprout Outgrowth Ex Vivo.

Carnosine, a naturally producing dipeptide, exhibits various beneficial effects. However, the possible role of carnosine in vascular disorders associated with pathological conditions, including proliferation and migration of vascular smooth muscle cells (VSMCs), largely remains unrevealed. Here, we investigated the regulatory role and mechanism of carnosine in platelet-derived growth factor (PDGF)-induced VSMCs. Carnosine inhibited the proliferation of PDGF-induced VSMCs without any cytotoxic effects. Carnosine treatment also induced G1-phase cell cycle arrest by causing a p21WAF1-mediated reduction in the expression of both cyclin-dependent kinases (CDKs) and cyclins in PDGF-treated VSMCs. Carnosine treatment suppressed c-Jun N-terminal kinase (JNK) phosphorylation in PDGF-stimulated signaling. Additionally, carnosine significantly prevented the migration of VSMCs exposed to PDGF. Carnosine abolished matrix metalloproteinase (MMP)-9 activity via reduced transcriptional binding activity of NF-κB, Sp-1, and AP-1 motifs in PDGF-treated VSMCs. Moreover, using aortic assay ex vivo, it was observed that carnosine addition attenuated PDGF-stimulated sprout outgrowth of VSMCs. Taken together, these results demonstrated that carnosine impeded the proliferation and migration of PDGF-stimulated VSMCs by regulating cell cycle machinery, JNK signaling, and transcription factor-mediated MMP-9 activity as well as prevented ex vivo sprout outgrowth of blood vessels. Thus, carnosine may be a potential candidate for preventing vascular proliferative disease.

Farrerol maintains the contractile phenotype of VSMCs via inactivating the extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase signaling.

Farrerol, a dihydroflavone isolated from Rhododendron dauricum L., can inhibit vascular smooth muscle cell (VSMC) proliferation and exert a protective effect on H2O2-induced vascular endothelial cells injury. In this study, we investigated the effects of farrerol on VSMC phenotypic modulation and balloon injury-induced vascular neointimal formation and explored the underlying mechanisms. Serum-starved rat thoracic aorta SMCs (RASMCs) were first pretreated with farrerol (3, 10, and 30μM, respectively), U0126 (a MEK kinase inhibitor), and SB203580 (a p38 kinase inhibitor), and followed by treatment with serum (10% FBS). The expression of several VSMC-specific markers, including α-SMA, SM22α, and OPN, were analyzed by western blot. Phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and p38 mitogen-activated protein kinase (MAPK) was also investigated. Farrerol inhibited the serum-induced transition of RASMCs from the contractile to the synthetic phenotype, and this was associated with a decrease in α-SMA and SM22α expression, and an increase in OPN expression. Farrerol also inhibited serum-induced phosphorylation of ERK1/2 and p38MAPK in RASMCs. Moreover, U0126 and SB203580 both inhibited the serum-induced phenotypic transition of RASMCs. These findings indicate that farrerol can maintain the contractile phenotype of VSMCs partly via inactivating the ERK1/2 and p38 MAPK signaling pathways. Using a rat model of carotid artery balloon injury, inhibition of VSMC phenotypic transition and suppression of neointimal formation were confirmed in vivo following the perivascular application of farrerol. Our results suggested that farrerol could be a promising lead compound for the treatment of vascular proliferative diseases.

Long Non-coding RNA PEBP1P2 Suppresses Proliferative VSMCs Phenotypic Switching and Proliferation in Atherosclerosis

Long non-coding RNAs (lncRNAs) play a crucial role in the growth of vascular smooth muscle cells (VSMCs), the dysfunction of which is closely associated with the initiation and progression of cardiovascular diseases (CVDs). Abnormal phenotypic switching and proliferation of VSMCs constitute a significant event in the progression of atherosclerosis. The present study identified a novel lncRNA, PEBP1P2, which serves as a valuable regulator of VSMCs in phenotypic transformation and proliferation. The expression of PEBP1P2 was remarkably decreased in proliferating VSMCs and pathological arteries when using a balloon injury model of rats. Furthermore, we found that PEBP1P2 represses proliferation, migration, and dedifferentiation during phenotype switching in VSMCs induced by platelet-derived growth factor BB (PDGF-BB). Mechanistically, cyclin-dependent kinase 9 (CDK9) was confirmed to be the direct target of PEBP1P2, which was proven to mediate phenotypic switching and proliferation of VSMCs and was rescued by PEBP1P2. Then, we explored the clinical significance, as we observed the decreased expression of PEBP1P2 in the serum of coronary heart disease (CHD) patients and human advanced carotid atherosclerotic plaques. Finally, PEBP1P2 overexpression distinctly suppressed neointima formation and VSMC phenotypic switching in vivo. Taken together, PEBP1P2 inhibits proliferation and migration in VSMCs by directly binding to CDK9, implying that it may be a promising therapeutic target for the treatment of proliferative vascular diseases.

Ruxolitinib attenuates intimal hyperplasia via inhibiting JAK2/STAT3 signaling pathway activation induced by PDGF-BB in vascular smooth muscle cells

BackgroundCardiovascular diseases are associated with proliferation and phenotypic switch. Platelet-derived growth factor-BB (PDGF-BB) is a major initiating factor for proliferative vascular diseases, such as neointimal lesion formation, restenosis after angioplasty, and atherosclerosis. Ruxolitinib, a potent Janus kinase (JAK) 1 and 2 inhibitor, has been reported to significantly block the proliferation-related signaling pathway of JAK2/signal transducers and activators of transcription 3 (STAT3) and harbor a broad spectrum of anti-cancer activities, including proliferation inhibition, apoptosis induction, and anti-inflammation. However, the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation remains to be elucidated. Thus, this study investigates the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation and its underlying mechanisms. MethodsIn vivo, the medial thickness of the carotid artery was evaluated using a mouse carotid ligation model, ruxolitinib was administered orally to the mice every other day, and the mice were euthanized on day 28 to evaluate the therapeutic effects of ruxolitinib. Cell proliferation markers were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. In vitro, VSMCs were treated with ruxolitinib with or without PDGF-BB at an indicated time and concentration. Cell proliferation and apoptosis were measured using Cell Counting Kit-8 assay, MTS assays and flow cytometry. The JAK2/STAT3 signaling pathway involved in the effects of ruxolitinib on VSMCs was detected by western blotting with the specific pathway inhibitor AG490. ResultsIn vivo, ruxolitinib significantly decreased the ratio-of-intima ratio (I/M ratio) by inhibiting the expression of PCNA and cyclinD1 (p <0.05). In vitro, ruxolitinib inhibited PDGF-BB-induced VSMC proliferation compared with the PDGF-BB treatment group (p <0.05). In addition, ruxolitinib inhibited the PDGF-BB-induced activation of the JAK2/STAT3 signaling pathway and decreased the expression of proliferation related-proteins cyclinD1 and PCNA in VSMCs (p <0.05). ConclusionOur findings suggest that ruxolitinib inhibits VSMC proliferation in vivo and in vitro by suppressing the activation of the JAK2/STAT3 signaling pathway. Therefore, ruxolitinib has a therapeutic potential for proliferative vascular diseases.

Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor-β.

Apatinib (YN968D1) is a small‐molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor‐2 (VEGFR‐2). It has been reported that apatinib has anti‐tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet‐derived growth factor type BB (PDGF‐BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor‐β (PDGFR‐β) and phosphoinositide‐specific phospholipase C‐γ1 (PLC‐γ1) induced by PDGF‐BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR‐β, extracellular signal‐related kinases (ERK1/2) and Jun amino‐terminal kinases (JNK) induced by PDGF‐BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF‐BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases.

Glycyrrhizic Acid Attenuates Balloon-Induced Vascular Injury Through Inactivation of RAGE Signaling Pathways

Percutaneous coronary intervention is a well-established technique used to treat coronary artery disease, but the risk of coronary artery in-stent restenosis following percutaneous coronary intervention is still high. Previous studies revealed that high mobility group protein B1 (HMGB1) plays a critical role in neointima formation. In this study, we aimed to investigate the role of glycyrrhizic acid (GA), an HMGB1 inhibitor, in the process of neointima formation and the potential mechanisms. We investigated the role of GA in neointima formation through an iliac artery balloon injury model in rabbits. Proliferation, migration, and phenotype transformation of human vascular smooth muscle cells (VSMCs) were observed. Besides, inflammation and receptor for advanced glycosylation end products (RAGE) signaling pathways were studied. The results indicate that GA attenuated neointima formation and downregulated HMGB1 expression in injured artery in rabbits. HMGB1 promoted proliferation, migration, and phenotype transformation through the activation of RAGE signaling pathways in VSMCs, and blockade of HMGB1 by GA (1, 10, and 100 μM) could attenuate those processes and reduce proliferation of human VSMCs. In conclusion, the HMGB1 inhibitor GA might be useful to treat proliferative vascular diseases by downregulating RAGE signaling pathways. Our results indicate a new and promising therapeutic agent for restenosis.

Tanshinone ⅡA inhibits VSMC inflammation and proliferation in vivo and in vitro by downregulating miR-712-5p expression

The inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the basic pathological feature of proliferative vascular diseases. Tanshinone ⅡA (Tan ⅡA), which is the most abundant fat-soluble element extracted from Salvia miltiorrhiza, has potent protective effects on the cardiovascular system. However, the underlying mechanism is still not fully understood. Here, we show that Tan ⅡA significantly inhibits neointimal formation and decreases VSMC inflammation by upregulating the expression of KLF4 and inhibiting the activation of NFκB signaling. Using a microRNA array analysis, we found that miR-712-5p expression is significantly upregulated in tumor necrosis factor alpha (TNF-α)-treated VSMCs. Loss- and gain-of-function experiments revealed that transfection of miR-712-5p mimic promotes, whereas depletion of miR-712-5p suppresses TNF-α-induced VSMC inflammation, leading to amelioration of intimal hyperplasia induced by carotid artery ligation. Moreover, depletion of miR-712-5p by its antagomir largely abrogates TNF-α-induced VSMC proliferation. Our findings suggest that miR-712-5p mediates the stimulatory effect of TNF-α on VSMC inflammation, and that Tan ⅡA inhibits VSMC inflammation and proliferation in vivo and in vitro by suppression of miR-712-5p expression. Targeting miR-712-5p may be a novel therapeutic strategy to prevent proliferative vascular diseases.

Endothelial Scaffolding Protein ENH (Enigma Homolog Protein) Promotes PHLPP2 (Pleckstrin Homology Domain and Leucine-Rich Repeat Protein Phosphatase 2)-Mediated Dephosphorylation of AKT1 and eNOS (Endothelial NO Synthase) Promoting Vascular Remodeling.

A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.

Evaluating peripheral retinal vascularization and rate of recurrence after intravitreal injection of ranibizumab as a primary treatment for retinopathy of prematurity

Context Retinopathy of prematurity (ROP) is a vascular proliferative disease affecting premature infants. Aims The aim was to assess the extent of peripheral retinal vascular growth and recurrence rate in patients with type 1 ROP treated with ranibizumab. Settings and design Patients with ROP attending the pediatric eye clinic in the main university hospital in Alexandria Faculty of Medicine were recruited. Participants and methods This prospective study included 20 eyes of 10 patients showing type 1 ROP, treated with 0.25 mg ranibizumab intravitreal injection only. Patients were followed for 6 months, where peripheral vascularization and rate of recurrence were assessed through indirect ophthalmoscopy and RetCam imaging. Statistical analysis Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. The tests used were χ2 test, Fisher’s exact test, and Student t test. Results A total of 16 (80%) eyes showed good peripheral vascularization and favorable anatomical outcome. The average timing for temporal zone III retinal vascularization was 15.71±2.09 weeks, with three eyes not reaching zone III after 6 months of injection. One eye showed retinal folding and macular ectopia. Three (15%) eyes showed signs of recurrence, with two eyes showing neovascularization at the initial vascular ridge by the fifth week after intravitreal ranibizumab injection (IVR), and one eye showing the recurrence of thick temporal ridge by the fourth months. Conclusions Ranibizumab is a potential effective treatment for patients with type 1 ROP. Delayed peripheral vascularization and disease recurrence mandate tailored long-term follow-up period according to individual response.

The course of delayed intrauterine infection in children with residual manifestations of retinopathy of prematurity

Retinopathy of prematurity (ROP) is a severe vascular proliferative disease. The appearance of signs of intrauterine infection in the setting of ROP may aggravate the severity of the disease course and lead to development of complications. Two children with residual ROP effects were observe to have delayed manifestations of intrauterine infection. The first child had signs of toxoplasmosis in the period of regression of stage II ROP at 48 weeks of postmenstrual age (PMA). The second child had signs of cytomegalovirus infection in the period of induced regression of III «+» stage ROP at 47-48 weeks of PMA. At the time of manifestation of intrauterine infection, both children were noted to have exudative phenomena, and formed fibrous bridles that caused retinal detachment, which was visually very similar to the late stages of severe forms of ROP. However, the appearance of these symptoms in 47-48 weeks of the PMA in the setting of ROP regress excludes the activation of the disease. The course of uveitis with residual manifestations of ROP went according to the most severe scenario - with retinal detachment in the short term - within 7-10 days.

261Decisive role of microRNA-494 in smooth muscle cell proliferation and vascular remodeling

Abstract Background Proliferation of vascular smooth muscle cells triggered by cytokines and growth factors is a main driver in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis after angioplasty. MicroRNAs (miR) are small noncoding RNAs that can inhibit the expression of multiple genes simultaneously. However, the contribution of microRNAs to the differential gene regulation that triggers vascular remodeling processes is not well understood. Methods and results Neointima formation was induced by a wire-mediated injury of the femoral artery in C57BL/6 mice. Microarray analysis of the developing neointimal lesion showed a strong reduction of miR-494 (0.411±0.04; p&lt;0.05) at 7 days after injury. In order to investigate the expression levels of miRs in vascular cells, human coronary artery smooth muscle cells (SMC), human coronary artery endothelial cells and human monocytes were analyzed via microarray analysis. Intriguingly, miR-494 was found to be predominantly expressed in SMC via microarray and qPCR analysis. The regulation of miR-494 expression was further analyzed after stimulation of SMC with 10%FCS. Following this mitogenic stimulation, mir-494 expression dropped robustly and significantly in a time-dependent manner at 6, 9, and 24 hours. To investigate the functional impact of miR-494 on SMC proliferation, miR-494 was overexpressed using miR-494-mimics (20μM). Overexpression of miR-494 significantly reduced the FCS-induced proliferation of SMC as assessed by BrdU-incorporation. In silico analyses of potential target genes for miR-494 identified ROCK1 and Survivin, both important molecules in the mitogenic response of SMC to cytokines and growth factors, as potential targets of miR-494. Indeed, ROCK1 and Survivin were found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and both mulecules could be identified as direct targets using luciferase reporter assays. Following the specific inhibition of miR-494 by local application (in a perivascular thermos-responsive, self-degrading pluronic gel) of an in vivo stabilized Pre-miR-494 after wire-induced injury of the mouse femoral artery, SMC proliferation was significantly reduced, as assessed by Ki67 immunofluorescence (26.3% vs 11.2%; p&lt;0.05). Consistently, local application of Pre-miR-494 significantly reduced neointima formation (neointima/media ratio 2.31 in control vs 1.01 in treated animals; p&lt;0.01). Conclusion Our results show that mir-494 is strongly down-regulated in proliferating SMC in vitro as well as during neointimal lesion formation in vivo. Moreover, overexpression/ reconstitution of miR-494 levels effectively prevented SMC proliferation and neointima formation, indicating an important functional role of miR-494 in these processes. Hence, miR-494 may represent an attractive SMC-specific target for future therapeutic interventions for the treatment of vascular proliferative diseases.

MicroRNA-125a-3p affects smooth muscle cell function in vascular stenosis

AimsMany studies have indicated that microRNAs are closely related to the process of peripheral arterial disease (PAD). Previously, we found that microRNA-125a-3p (miR-125a-3p) in restenotic arteries after interventional therapy of lower extremity vessels was notably decreased compared with that of normal control arteries. However, its role in the development of vascular stenosis is not yet clearly understood. The purpose of this study was to investigate the expression, regulatory mechanism and function of miR-125a-3p in the process of vascular stenosis. Methods and resultsQuantitative reverse-transcription polymerase chain reaction assays indicated that miR-125a-3p in restenotic arteries after interventional therapy was significantly lower than that in normal control arteries. Immunofluorescence and in situ hybridization co-staining assays in arterial sections demonstrated that miR-125a-3p was mainly expressed in the medial smooth muscle layer. Transfection of miR-125a-3p mimics into cultured vascular smooth muscle cells (VSMCs) effectively inhibited cell proliferation and migration. Then, western blot and luciferase activity assays showed that recombinant human mitogen-activated protein kinase 1 (MAPK1) was a functional target of miR-125a-3p and was involved in miR-125a-3p-mediated cell effects. Finally, the lentiviral infection of miR-125a-3p in balloon-injured rat carotid vascular walls showed that miR-125a-3p overexpression significantly reduced the probability of neointimal membrane production. ConclusionsmiR-125a-3p can effectively inhibit the function of VSMCs and the occurrence of vascular stenosis by targeting MAPK1. This study introduces a new molecular mechanism of PAD. We show that regulation of the miR-125a-3p level has the potential to provide a new treatment for PAD and other proliferative vascular diseases.

Hyperglycaemia as a risk factor for the development of retinopathy of prematurity: A cohort study

BackgroundRetinopathy of prematurity (ROP) is a preventable cause of childhood blindness. Without treatment, over 45% of eyes can develop permanent visual loss. Hyperglycaemia has recently been described as a risk factor for the development of retinopathy of prematurity (ROP), a proliferative vascular disease of the retina that primarily affects premature infants. The characteristic neoproliferative growth of blood vessels in the retina is very well under stood with the clinical and experimental experiences with Diabetic retinopathy. The purpose of this study was to evaluate a possible relation between glucose levels in VLBW (Very Low Birth Weight) infants and development of ROP. MethodAll at risk infants of a Neonatal Intensive Care Unit (NICU) of a tertiary care centre in western India were included in the study. The blood sugar values of the neonates were recorded at multiple times during their first week of life. On completion of 31 weeks of gestational age or 04 weeks of birth age, the neonates were subjected to ROP screening as per standard protocols. ResultA total of 103 neonates were included in the study and were subjected to ROP screening. A total of 32 neonates developed ROP at the end of the study. It was found with statistical significance that the neonates with higher average blood glucose values in the initial period of life had higher incidence of ROP at the time of screening with a Relative Risk of 2.506 (CI = 1.287, 4.882). ConclusionA high average blood glucose level in neonates during the first week of life is an indicator for developing ROP at a later date. These neonates should be kept under close follow up in order to facilitate timely detection and prompt intervention.

Association between low levels of serum miR-638 and atherosclerotic plaque vulnerability in patients with high-grade carotid stenosis.

Carotid artery atherosclerosis is a major cause of ischemic stroke. However, reliable criteria to identify patients with high-risk carotid plaques beyond the severity of stenosis are still lacking. Circulating microRNAs (miRNAs) are being postulated as biomarkers for a variety of vascular immune-inflammatory diseases. The authors investigated whether cell-free circulating miR-638, highly expressed in vascular smooth muscle cells and implicated in proliferative vascular diseases, is associated with vulnerable atherosclerotic plaques in high-risk patients with advanced carotid artery stenosis undergoing carotid endarterectomy (CEA). The authors conducted a prospective study in 22 consecutive symptomatic patients with high-grade carotid stenosis undergoing CEA and 36 age- and sex-matched patients without ischemic stroke history or carotid atherosclerosis (control group). In addition, they reviewed data from a historical group of 9 CEA patients who underwent long-term follow-up after revascularization. Total RNA was isolated from all serum samples, and relative miR-638 expression levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and compared among groups. A correlation analysis of serum miR-638 levels with vascular risk factors and treatments, and with plaque features, was performed. The ability of serum miR-638 to discriminate between the non-CEA control group and the different CEA groups was assessed by receiver operating characteristic evaluation. A logistic regression model was employed to examine the association between stratified CEA patients and serum miR-638 levels. Serum levels of miR-638 were significantly lower in symptomatic CEA patients (p = 0.009) and particularly in the subgroup of CEA patients who had experienced stroke (p = 0.0006) than in non-CEA controls. Discrimination of high-risk plaques was accurate (area under the curve [AUC] 0.66 for symptomatic CEA patients in general and 0.76 for those who had experienced stroke). When only patients with high cardiovascular risk were considered, the diagnostic value of serum miR-638 from symptomatic CEA patients and CEA patients who had experienced stroke improved (AUC 0.79 and 0.85). Moreover, serum miR-638 was negatively correlated with the occurrence of stroke, smoker status, presence of bilateral pathology, coronary artery disease, and cholesterol treatment; and with the high-risk fibroatheroma plaques extracted from CEA patients. Multivariate logistic regression analysis demonstrated that serum miR-638 was an independent predictor of plaque instability. Furthermore, serum miR-638 appeared to attain good discrimination for atherosclerotic stenosis in CEA patients based on analysis of blood samples obtained in the historical group before and 5 years after intervention (p = 0.04) (AUC = 0.79). According to this preliminary proof-of-concept study, serum miR-638 might constitute a promising noninvasive biomarker associated with plaque vulnerability and ischemic stroke, particularly in individuals with elevated cardiovascular risk.