Pathological Vascular Smooth Muscle Cell Research Articles

Lipoprotein(a) induces spontaneous aortic dissection via increasing monocyte mediated vascular smooth muscle cell phenotype transformation in human Lp(a) transgenic mice

Abstract Background Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent risk factor of atherosclerotic cardiovascular disease and aortic valve stenosis. Recent studies have reported that high levels of Lp(a) are strongly associated with aortic dissection (AD), independent of other cardiovascular risk factors. However, orthologues of Lp(a) are only found in humans, Old World monkeys, apes, and hedgehogs. The lack of a proper animal model has hindered elucidation of the true pathophysiological mechanisms of Lp(a). Here, we conducted the first humanized Lp(a) transgenic mice experiment to verify the pathological mechanism of Lp(a) in aortic dissection. Methods We conducted ApoE knock out and human ApoA transgenic mice via CRISPR-Cas9 and then crossed with human ApoB transgenic mice to achieve high levels of human-like Lp(a) particles in plasma. Plasma was assayed by ELISA for the concentrations of each lipoprotein. Echocardiography was performed to detect aortic morphology in vivo. Single cell transcriptome analysis was performed on the dissection lesion. Cell interaction was examined in macrophage from Lp(a) transgenic mice and vascular smooth muscle cell (VSMC) coculture system. Result Our results indicate that the concentration of ApoA and ApoB in plasma were increased in transgenic mice fed a chow diet. Low density lipoprotein (LDL) levels were increased and shows that LDL is the major cholesterol carrier in these mice. We were surprised to find that the dissection of the ascending aortic arch could be seen by echocardiography in transgenic mice as early as 2 months, and transgenic mice showed significant increase in aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions, resulting in a survival rate of only 58% after 12 months. Single cell transcriptome analysis revealed that Lp(a) transgene induced the inflammatory response, dedifferentiation and pathological VSMC phenotypical switch, and matrix metalloproteinase expression in macrophages. Monocytes isolated from transgenic mice with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation. Monocytes isolated from wild type mice cultured in media with high level Lp(a) for 24h also showed the ability to facilitated cocultured VSMC phenotype transformation, which was reversed by E06, a monoclonal antibody blocking the oxidized phospholipids in Lp(a). Conclusion These findings demonstrate that Lp(a) induces spontaneous aortic dissection via increasing monocyte trafficking to the arterial wall, mediating proinflammatory responses and VSMC phenotype transformation through its OxPL content. The study presents a rodent model of spontaneous aortic dissection and provide a novel mechanism by which Lp(a) mediates aortic dissection.Graphical abstract

Glycolysis and de novo fatty acid synthesis cooperatively regulate pathological vascular smooth muscle cell phenotypic switching and neointimal hyperplasia.

Switching of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a dedifferentiated (proliferative) phenotype contributes to neointima formation, which has been demonstrated to possess a tumor-like nature. Dysregulated glucose and lipid metabolism is recognized as a hallmark of tumors but has not thoroughly been elucidated in neointima formation. Here, we investigated the cooperative role of glycolysis and fatty acid synthesis in vascular injury-induced VSMC dedifferentiation and neointima formation. We found that the expression of hypoxia-inducible factor-1α (HIF-1α) and its target 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a critical glycolytic enzyme, were induced in the neointimal VSMCs of human stenotic carotid arteries and wire-injured mouse carotid arteries. HIF-1α overexpression led to elevated glycolysis and resulted in a decreased contractile phenotype while promoting VSMC proliferation and activation of the mechanistic target of rapamycin complex 1 (mTORC1). Conversely, silencing Pfkfb3 had the opposite effects. Mechanistic studies demonstrated that glycolysis generates acetyl coenzyme A to fuel de novo fatty acid synthesis and mTORC1 activation. Whole-transcriptome sequencing analysis confirmed the increased expression of PFKFB3 and fatty acid synthetase (FASN) in dedifferentiated VSMCs. More importantly, FASN upregulation was observed in neointimal VSMCs of human stenotic carotid arteries. Finally, interfering with PFKFB3 or FASN suppressed vascular injury-induced mTORC1 activation, VSMC dedifferentiation, and neointima formation. Together, this study demonstrated that PFKFB3-mediated glycolytic reprogramming and FASN-mediated lipid metabolic reprogramming are distinctive features of VSMC phenotypic switching and could be potential therapeutic targets for treating vascular diseases with neointima formation. © 2023 The Pathological Society of Great Britain and Ireland.

Microskeletal stiffness promotes aortic aneurysm by sustaining pathological vascular smooth muscle cell mechanosensation via Piezo1

Mechanical overload of the vascular wall is a pathological hallmark of life-threatening abdominal aortic aneurysms (AAA). However, how this mechanical stress resonates at the unicellular level of vascular smooth muscle cells (VSMC) is undefined. Here we show defective mechano-phenotype signatures of VSMC in AAA measured with ultrasound tweezers-based micromechanical system and single-cell RNA sequencing technique. Theoretical modelling predicts that cytoskeleton alterations fuel cell membrane tension of VSMC, thereby modulating their mechanoallostatic responses which are validated by live micromechanical measurements. Mechanistically, VSMC gradually adopt a mechanically solid-like state by upregulating cytoskeleton crosslinker, α-actinin2, in the presence of AAA-promoting signal, Netrin-1, thereby directly powering the activity of mechanosensory ion channel Piezo1. Inhibition of Piezo1 prevents mice from developing AAA by alleviating pathological vascular remodeling. Our findings demonstrate that deviations of mechanosensation behaviors of VSMC is detrimental for AAA and identifies Piezo1 as a novel culprit of mechanically fatigued aorta in AAA.

Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch.

Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP–treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype–associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.

LncRNA AC105942.1 Downregulates hnRNPA2/B1 to Attenuate Vascular Smooth Muscle Cells Proliferation.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) is crucial in the atherosclerosis. Although long noncoding RNAs (lncRNAs) are implicated in a variety of diseases, their roles in activation of VSMCs proliferation and vascular disorder diseases are not well understood. In addition, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) was reported to participate in lncRNAs-mediated function. Herein, we propose to investigate the role of lncRNA AC105942.1 and hnRNPA2/B1 in pathological VSMCs proliferation and the possible mechanisms in vitro. We have identified that lncRNA AC105942.1 was downregulated and hnRNPA2/B1 was upregulated in atherosclerotic plaques compared with normal artery tissues. Enhanced lncRNA AC105942.1 could noticeably inhibit Ang II-induced VSMCs proliferation. Further investigation suggested that lncRNA AC105942.1 could downregulate the expression of hnRNPA2/B1 and then regulate the level of CDK4 and p27. Taken together, our study indicated that lncRNA AC105942.1 downregulated hnRNPA2B1 to protect against the atherosclerosis by suppressing VSMCs proliferation. LncRNA AC105942.1 and hnRNPA2/B1 could represent potential therapeutic and diagnostic targets to atherosclerosis-related diseases.

Prevention of aortic dissection and aneurysm via an ALDH2-mediated switch in vascular smooth muscle cell phenotype.

Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive. Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs. ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.

MiR‐92b‐3p‐TSC1 axis is critical for mTOR signaling‐mediated vascular smooth muscle cell proliferation induced by hypoxia

Pulmonary artery smooth muscle cells (PASMCs) undergo proliferation by the mammalian target of rapamycin (mTOR) signaling pathway under hypoxia. Hypoxia induces expression of a specific set of microRNAs (miRNAs) in a variety of cell types. We integrated genomic analyses of both small non‐coding RNA and coding transcripts using next‐generation sequencing (NGS)‐based RNA sequencing with the molecular mechanism of the mTOR signaling pathway in hypoxic PASMCs. These analyses revealed hypoxia‐induced miR‐92b‐3p as a potent regulator of the mTOR signaling pathway. We demonstrated that miR‐92b‐3p directly targets the 3′ UTR of a negative regulator in the mTOR signaling pathway, TSC1. mTOR signaling and consequent cell proliferation were promoted by enforced expression of miR‐92b‐3p but inhibited by knocking down endogenous miR‐92b‐3p. Furthermore, inhibition of miR‐92b‐3p attenuated hypoxia‐induced proliferation of vascular smooth muscle cells (VSMCs). Therefore, this study elucidates a novel role of miR‐92b‐3p as a hypoxamir in the regulation of the mTOR signaling pathway and the pathological VSMC proliferative response under hypoxia. These findings will help us better understand the miRNA‐mediated molecular mechanism of the proliferative response of hypoxic VSMCs through the mTOR signaling pathway.

Survivin is a Mechanosensitive Regulator of Vascular Smooth Muscle Cell Proliferation

Arterial stiffness induces pathological vascular smooth muscle cell (VSMC) phenotypes, causing atherogenesis and arterial stenosis when rigid arterial extracellular matrix signals VSMCs to aberrantly proliferate. Survivin is an inhibitor of apoptosis but alternatively promotes cell proliferation, and mounting evidence points to survivin inhabiting a key role in vascular injury and stiffening responses. Thus, we seek to understand if survivin plays a role in how mechanical forces drive the pathological proliferation of VSMCs.

Canonical Transient Receptor Potential Channels and Vascular Smooth Muscle Cell Plasticity.

Vascular smooth muscle cells (VSMCs) play a pivotal role in the stability and tonic regulation of vascular homeostasis. VSMCs can switch back and forth between highly proliferative (synthetic) and fully differentiated (contractile) phenotypes in response to changes in the vessel environment. Abnormal phenotypic switching of VSMCs is a distinctive characteristic of vascular disorders, including atherosclerosis, pulmonary hypertension, stroke, and peripheral artery disease; however, how the control of VSMC phenotypic switching is dysregulated under pathological conditions remains obscure. Canonical transient receptor potential (TRPC) channels have attracted attention as a key regulator of pathological phenotype switching in VSMCs. Several TRPC subfamily member proteins—especially TRPC1 and TRPC6—are upregulated in pathological VSMCs, and pharmacological inhibition of TRPC channel activity has been reported to improve hypertensive vascular remodeling in rodents. This review summarizes the current understanding of the role of TRPC channels in cardiovascular plasticity, including our recent finding that TRPC6 participates in aberrant VSMC phenotype switching under ischemic conditions, and discusses the therapeutic potential of TRPC channels.

Suv39h1 downregulation inhibits neointimal hyperplasia after vascular injury

Background and aimsNeointimal hyperplasia resulting from pathological vascular smooth muscle cells (VSMCs) activation is a common pathophysiological basis for numerous proliferative vascular diseases, such as restenosis. Suv39h1, an important transcription suppressor, may be involved in this process. Herein, we investigated the role of Suv39h1 in pathological intimal hyperplasia and its possible mechanisms in vitro and in vivo. MethodsAn adenovirus vector for Suv39h1 overexpression and a lentiviral vector for its downregulation were constructed and used to transfect cultured VSMCs in vitro. The functional changes in VSMCs stimulated by angiotensin II (Ang II) were observed and the possible mechanism was investigated. Additionally, rat carotid arteries with balloon injury were locally transfected with these viral vectors and changes in neointima formation, proliferating cell nuclear antigen (Pcna) expression and collagen deposition were examined. ResultsUpon Ang II stimulation, the expression of Suv39h1 and inhibitor of DNA binding 3 (Id3) was significantly increased. Suv39h1 downregulation inhibited Ang II-stimulated migration and proliferation of VSMCs, antagonized the production of Id3 and promoted p21 and p27Kip1 expression. In contrast, Suv39h1 overexpression had the opposite effects. Suv39h1 regulated the transcription of p21 and p27Kip1 by controlling H3K9me3 in the proximal promoter regions. Consistent with the VSMCs results, Suv39h1 and Id3 expression was significantly increased in blood vessels after balloon injury. Suv39h1 downregulation inhibited intimal hyperplasia, and attenuated Pcna expression and collagen synthesis in the intima, while Suv39h1 overexpression had the opposite effects. ConclusionsSuv39h1 downregulation effectively inhibited neointimal hyperplasia after vascular injury.

Smooth muscle cell-specific knockout of neuropilin-1 impairs postnatal lung development and pathological vascular smooth muscle cell accumulation.

Neuropilin 1 (NRP1) is important for neuronal and cardiovascular development due to its role in conveying class 3 semaphorin and vascular endothelial growth factor signaling, respectively. NRP1 is expressed in smooth muscle cells (SMCs) and mediates their migration and proliferation in cell culture and is implicated in pathological SMC remodeling in vivo. To address the importance of Nrp1 for SMC function during development, we generated conditional inducible Nrp1 SMC-specific knockout mice. Induction of early postnatal SMC-specific Nrp1 knockout led to pulmonary hemorrhage associated with defects in alveogenesis and revealed a specific requirement for Nrp1 in myofibroblast recruitment to the alveolar septae and PDGF-AA-induced migration in vitro. Furthermore, SMC-specific Nrp1 knockout inhibited PDGF-BB-stimulated SMC outgrowth ex vivo in aortic ring assays and reduced pathological arterial neointima formation in vivo. In contrast, we observed little significant effect of SMC-specific Nrp1 knockout on neonatal retinal vascularization. Our results point to a requirement of Nrp1 in vascular smooth muscle and myofibroblast function in vivo, which may have relevance for postnatal lung development and for pathologies characterized by excessive SMC and/or myofibroblast proliferation.

MiR-92b-3p-TSC1 axis is critical for mTOR signaling-mediated vascular smooth muscle cell proliferation induced by hypoxia

Pulmonary artery smooth muscle cells (PASMCs) undergo proliferation by the mammalian target of rapamycin (mTOR) signaling pathway under hypoxia. Hypoxia induces expression of a specific set of microRNAs (miRNAs) in a variety of cell types. We integrated genomic analyses of both small non-coding RNA and coding transcripts using next-generation sequencing (NGS)-based RNA sequencing with the molecular mechanism of the mTOR signaling pathway in hypoxic PASMCs. These analyses revealed hypoxia-induced miR-92b-3p as a potent regulator of the mTOR signaling pathway. We demonstrated that miR-92b-3p directly targets the 3′-UTR of a negative regulator in the mTOR signaling pathway, TSC1. mTOR signaling and consequent cell proliferation were promoted by enforced expression of miR-92b-3p but inhibited by knocking down endogenous miR-92b-3p. Furthermore, inhibition of miR-92b-3p attenuated hypoxia-induced proliferation of vascular smooth muscle cells (VSMCs). Therefore, this study elucidates a novel role of miR-92b-3p as a hypoxamir in the regulation of the mTOR signaling pathway and the pathological VSMC proliferative response under hypoxia. These findings will help us better understand the miRNA-mediated molecular mechanism of the proliferative response of hypoxic VSMCs through the mTOR signaling pathway.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

P12 Pathological vascular smooth muscle cells remodelling in murine models of abdominal aortic aneurysm

Rationale Abdominal aortic aneurysm (AAA) is a focal dilation of the abdominal aorta that progresses to rupture, which is frequently fatal. The only existing treatments are high risk surgical interventions. These are not offered until the AAA is large (>5.5?cm) so patients remain under surveillance for many years. There is currently no medical therapy to offer. We have previously observed a senescent phenotype in vascular smooth muscle cells (VSMC) isolated from end-stage human AAA. We hypothesise that there is a prior period of pathological VSMC remodelling which may be a promising and novel target for molecular therapies. Methodology Three murine models of early AAA were used – (i) AngII infusion to ApoE-/- mice and (ii) CaCl2 or (iii) porcine pancreatic elastase application to the aortic adventitia of C57BL6/J mice. VSMC remodelling was investigated in fixed and paraffin embedded aortic tissue harvested from the murine models using the histochemical stain Movat’s Pentachrome and immunohistochemistry for the VSMC identification marker, αSMA. Slides were imaged with a digital slide scanner or confocal microscopy as appropriate. Aortic lumen volume was determined by in-vivo ultrasound. Results We observed VSMC remodelling in all three models of AAA. The remodelling was reduced by administration of a novel small-molecule inhibitor of PDGF induced VSMC remodelling. Inhibition of VSMC remodelling also reduced the severity of the aneurysms formed as assessed by 3D in-vivo ultrasound of the aortic lumen. Conclusions There is a major VSMC remodelling response in murine AAA and disrupting this response appears to suppress AAA formation.

Nucleoside diphosphate kinase B-activated intermediate conductance potassium channels are critical for neointima formation in mouse carotid arteries.

Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca(2+)-activated K(+) (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (ISK4) in proliferating VSMC and neointima formation. Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased ISK4, whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in ISK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

61. Vascular Smooth Muscle Cell RNA Aptamers for the Treatment of Cardiovascular Disease

Cardiovascular disease (CVD) is the leading cause of mortality in many countries. Many vascular disorders, including in-stent restenosis, arteriosclerosis, vein graft disease, and cardiac allograft arteriopathy are caused by pathological vascular smooth muscle cell (VSMC) remodeling following injury. An ideal therapeutic intervention would target the VSMCs without impairing the injured vessel re-endothelialization. However, current therapies do not selectively prevent pathological VSMC remodeling leading to impaired re-endothelization, late stent thrombosis and death. Thus, there is a clear need for cell-targeted treatment and prevention options of pathological VSMC remodeling.Our group has described the development of VSMC-specific, aptamers for (1) modulating signaling pathways associated with pathological VSMC remodeling and (2) delivering therapeutic molecules to these cells in vivo. Here we demonstrate that one of these aptamers, Vapt14, inhibits protein kinase B (PKB)/Akt activation and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-beta phosphorylation. In a murine model of carotid injury, treatment of vessels with Vapt14 reduces intimal:medial thickness to levels comparable to that of paclitaxel. Importantly, we confirm that Vapt14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells (PBMCs) in vitro. In addition, we confirm delivery of Vapt14 to VSMC in vitro and in vivo with fluorescence microscopy. Studies are being expanded to evaluate aptamer-mediated delivery of therapeutic biomolecules (e.g. small molecules, RNAi modulators) to areas of vascular injury. In summary this work provides an essential foundation for the translation of cell-targeted RNA therapeutics to multiple hyperplastic vascular diseases.

RACing up a New Regulatory Mechanism for Vascular Smooth Muscle Cell Migration

Pathophysiological vascular smooth muscle cell (VSMC) migration is a critical component of atherosclerosis and contributes substantially to neointimal hyperplasia. Injury to the intimal layer of the vessel promotes the dedifferentiation of VSMCs in the tunica media from a quiescent contractile phenotype to a synthetic phenotype that proliferates and migrates, thus contributing to neointimal thickening. A better understanding of the signaling mechanisms that promote VSMC dedifferentiation, proliferation, and migration may lead to the identification of new pharmacological targets for atherosclerosis and other vascular diseases. See accompanying article on page 702 Several signal transduction pathways regulate actin polymerization and cell contractility, both integral to VSMC migration. The process of cell migration is complex but requires fundamental processes that depend on tight regulation of the GTPases Rac and Rho (Figure).1–3 Migration initiates when a cell is exposed to a chemoattractant gradient and establishes polarity. This triggers the extension of the plasma membrane, called a lamellipodium, in the direction of eventual cell movement and establishes the front/leading edge of the cell.2,4 In vascular injury, a gradient is often established by the release of platelet-derived growth factor (PDGF), such that VSMCs migrate toward the lumen of the vessel.4,5 The formation of lamellipodia requires the polymerization and assembly of actin, which is regulated by the GTPase Rac. …

MiR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat

Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.

Testosterone Attenuates Matrix Metalloproteinase Activity and the Cellular Processes of Intimal Hyperplasia In Vitro

Low testosterone levels have been associated with increased risk for vascular disease. Previous studies have shown a significant decrease in intimal hyperplasia (IH) development in the presence of testosterone. Matrix metalloproteinases (MMPs) play a major role in vascular remodeling due to their ability to selectively degrade components of the extracellular matrix, allowing pathological vascular smooth muscle cell (VSMC) migration and proliferation to the intimal layer. Our group has previously shown a positive correlation between estrogen/progesterone and IH development via increased MMP activity in vitro and in vivo.

Prediction and prevention by progenitors? Stent thrombosis and EPCs

This editorial refers to ‘Circulating endothelial progenitor cell levels and function in patients who experienced late coronary stent thrombosis’[†][1], by E.I. Lev et al. , on page 2625 During the late 1980s balloon-expandable vascular stents were developed to prevent abrupt closure and restenosis after angioplasty.1 Their initial success was hampered by an overwhelming rate of early stent thrombosis. Introduction of P2Y-receptor antagonists (i.e. ticlopidine and clopidogrel) for platelet inhibition in combination with acetylsalicylic acid greatly reduced this often fatal complication. Large long-term clinical studies, however, identified in-stent restenosis as an additional hindrance of sustained success. New drug-eluting stents (DES) promised to inhibit the pathological vascular smooth muscle cell migration and proliferation and proteoglycan deposition responsible for restenosis. To the dismay of all of us, the reduced risk of restenosis with DES was accompanied by an increased stent thrombosis rate. Unlike stent thrombosis after implantation of a bare metal stent, which generally occurs within the first 2 weeks, late (>30 days) and very late (>1 months) stent thrombosis is not infrequent after implantation of a DES.2 The main contributors leading to stent thrombosis include procedure-related factors such as stent diameter and length, apposition or expansion, patient-related factors including reduced ejection fraction, diabetes mellitus, or advanced age, antiplatelet-related factors comprising different drugs and individual responsiveness, stent thrombogenicity-related factors incorporating strut thickness, polymer type, and polymer thickness, and, most importantly, local and systemic factors influencing endothelial integrity, activation, and re-endothelialization.3 In in-stent thrombus formation, the initial tethering of platelets is activated by free stent struts and extracellular matrix proteins. It is mediated by the glycoprotein Ib/V/IX receptor complex and its major ligand von Willebrand factor.4 After the initial platelet adhesion, autocrine and paracrine mediators, including adenosine diphosphate (ADP), thrombin, epinephrine, and thromboxane A2, amplify and sustain the platelet response by … *Corresponding author. Tel: +49 228 2871 5217/5218, Fax: +49 228 2871 6423, Email: georg.nickenig{at}ukb.uni-bonn.de [1]: #fn-2