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Aims Recent studies revealed that the bromodomain and extra-terminal (BET) epigenetic reader proteins resemble key regulators in the underlying pathophysiology of cancer, diabetes, or cardiovascular disease. However, whether they also regulate vascular remodelling processes by direct effects on vascular cells is unknown. In this study, we investigated the effects of the BET proteins on human smooth muscle cell (SMC) function in vitro and neointima formation in response to vascular injury in vivo. Methods and results Selective inhibition of BETs by the small molecule (+)-JQ1 dose-dependently reduced proliferation and migration of SMCs without apoptotic or toxic effects. Flow cytometric analysis revealed a cell cycle arrest in the G0/G1 phase in the presence of (+)-JQ1. Microarray- and pathway analyses revealed a substantial transcriptional regulation of gene sets controlled by the Forkhead box O (FOXO1)1-transcription factor. Silencing of the most significantly regulated FOXO1-dependent gene, CDKN1A, abolished the antiproliferative effects. Immunohistochemical colocalization, co-immunoprecipitation, and promoter-binding ELISA assay data confirmed that the BET protein BRD4 directly binds to FOXO1 and regulates FOXO1 transactivational capacity. In vivo, local application of (+)-JQ1 significantly attenuated SMC proliferation and neointimal lesion formation following wire-induced injury of the femoral artery in C57BL/6 mice. Conclusion Inhibition of the BET-containing protein BRD4 after vascular injury by (+)-JQ1 restores FOXO1 transactivational activity, subsequent CDKN1A expression, cell cycle arrest and thus prevents SMC proliferation in vitro and neointima formation in vivo. Inhibition of BET epigenetic reader proteins might thus represent a promising therapeutic strategy to prevent adverse vascular remodelling.

BMJ open 12(4), e050067 (2022). doi:10.1136/bmjopen-2021-050067, Published by BMJ Publishing Group, London

SummaryCleaved high-molecular-weight kininogen (HKa) or its peptide domain 5 (D5) alone exert anti-adhesive properties in vitro related to impeding integrin-mediated cellular interactions. However, the anti-adhesive effects of HKa in vivo remain elusive. In this study, we investigated the effects of HKa on leukocyte recruitment and neointima formation following wire-induced injury of the femoral artery in C57BL/6 mice. Local application of HKa significantly reduced the accumulation of monocytes and also reduced neointimal lesion size 14 days after injury. Moreover, C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein (eGFP) showed a significantly reduced accumulation of eGFP+-cells at the arterial injury site and decreased neointimal lesion size after local application of HKa or the polypeptide D5 alone. A differentiation of accumulating eGFP+-cells into highly specific smooth muscle cells (SMC) was not detected in any group. In contrast, application of HKa significantly reduced the proliferation of locally derived neointimal cells. In vitro, HKa and D5 potently inhibited the adhesion of SMC to vitronectin, thus impairing their proliferation, migration, and survival rates. In conclusion, application of HKa or D5 decreases the inflammatory response to vascular injury and exerts direct effects on SMC by impeding the binding of integrins to extracellular matrix components. Therefore, HKa and D5 may hold promise as novel therapeutic substances to prevent neointima formation.

Objective— Bone marrow-derived progenitor cells have been implicated to contribute to neointima formation, but the time course and extent of their accumulation and differentiation into vascular cells and, most importantly, the long-term contribution of bone marrow-derived progenitor cells to the vascular lesion remain undefined. Methods and Results— Wire-induced injury of the femoral artery was performed on chimeric C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein, and vessels were harvested at 3 days, 1, 2, 3, 4, 6, and 16 weeks after dilatation (n=8 animals per time point). Using high-resolution microscopy, we unexpectedly found that the expression of smooth muscle cell or endothelial cell markers in enhanced green fluorescence protein positive cells was a very rare event. Indeed, most of the enhanced green fluorescence protein positive cells that accumulated during the acute inflammatory response were identified as monocytes/macrophages, and their number declined at later time points. In contrast, a substantial fraction of highly proliferative stem cell antigen-1 and CD34 + but enhanced green fluorescence protein negative and thus locally derived cells were detected in the adventitia. Conclusion— These data provide evidence that the differentiation of bone marrow-derived progenitor cells into smooth muscle cell or endothelial cell lineages seems to be an exceedingly rare event. Moreover, the contribution of bone marrow-derived cells to the cellular compartment of the neointima is limited to a transient period of the inflammatory response.

Various virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)–mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3−/− mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

Background Factor VII-activating protease (FSAP) is a multifunctional circulating plasma serine protease involved in thrombosis and vascular remodeling processes. The Marburg I single-nucleotide polymorphism (MI-SNP) in the FSAP-coding gene is characterized by low proteolytic activity, and is associated with increased rates of stroke and carotid stenosis in humans. Objectives To determine whether neointima formation after vascular injury is increased in FSAP(-/-) mice. Methods and Results The neointimal lesion size and the proliferation of vascular smooth muscle cells (VSMCs) were significantly enhanced in FSAP(-/-) mice as compared with C57BL/6 control mice after wire-induced injury of the femoral artery. Accumulation of leukocytes and macrophages was increased within the lesions of FSAP(-/-) mice at day 3 and day 14. Quantitative zymography demonstrated enhanced activity of gelatinases/matrix metalloproteinase (MMP)-2 and MMP-9 within the neointimal lesions of FSAP(-/-) mice, and immunohistochemistry showed particular costaining of MMP-9 with accumulating leukocytes. Using intravital microscopy, we observed that FSAP deficiency promoted the intravascular adherence and the subsequent transmigration of leukocytes in vivo in response to chemokine ligand 2 (CCL2). CCL2 expression was increased in FSAP(-/-) monocytes but not in the vessel wall. There was no difference in the expression of platelet-derived growth factor (PDGF-BB). Conclusions FSAP deficiency causes an increase in CCL2 expression and CCL2-mediated infiltration of leukocytes into the injured vessel, thereby promoting SMC proliferation and migration by the activation of leukocyte-derived gelatinases. These results provide a possible explanation for the observed association of the loss-of-function MI-SNP with vascular proliferative diseases.

The G534E polymorphism (Marburg I [MI]) of factor VII–activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Introduction: Proliferation of vascular smooth muscle cells (VSMC) plays a crucial role in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis. MicroRNAs (miR) are small noncoding RNAs that modulate gene expression in a posttranscriptional manner. Hypothesis: in this project we aimed to determine the functional impact of a micro-RNA which is strongly regulated during vascular remodeling. 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) at 7 days after injury as compared to uninjured controls, which could be confirmed by qPCR. Interestingly, miR-494 was found to be predominantly expressed in SMC as determined by in situ hybridization. The regulation of miR-494 expression was further analyzed in vitro 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 and total cell counts. In silico analyses of potential target genes for miR-494 identified survivin, an important molecule for cell cycle regulation, as potential target of miR-494. Indeed, survivin was found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and could be confirmed as direct target using luciferase reporter assays. Conclusions: Taken together, 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, the anti-proliferative effect of miR-494-reconstitution reveals a crucial functional role of miR-494 in the mitogenic response of SMC. Thus, miR-494 might represent a novel and promising SMC-specific target for future therapeutic strategies in the treatment of vascular proliferative diseases.

Aims MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial- specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice. Methods and results MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin a5 and sirtuin1, and reduced eNOS expression in vitro. MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo, systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotypewas observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin a5 and sirtuin1. Conclusion Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury.

Aims Being central part of the DNA repair machinery, DNA-dependent protein kinase (DNA-PK) seems to be involved in other signalling processes, as well. NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (SMC) proliferation and in vascular proliferative processes. We determined putative phosphorylation sites of NDA-PK in NOR1 and hypothesized that the enzyme is able to modulate NOR1 signalling and, this way, proliferation of SMC. Methods and results Cultured human aortic SMC were treated with the specific DNA-PK inhibitor NU7026 (or siRNA), which resulted in a 70% inhibition of FCS-induced proliferation as measured by BrdU incorporation. Furthermore, FCS-stimulated up-regulation of NOR1 protein as well as the cell-cycle promoting proteins proliferating cell nuclear antigen (PCNA), cyclin D1, and hyperphosphorylation of the retinoblastoma protein were prevented by DNA-PK inhibition. Co-immunoprecipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR1. Mutational analysis and kinase assays demonstrated that NOR1 is a substrate of DNA-PK and is phosphorylated in the N-terminal domain. Phosphorylation resulted in post-transcriptional stabilization of the protein through prevention of its ubiquitination. Active DNA-PK and NOR1 were found predominantly expressed within the neointima of human atherosclerotic tissue specimens. In mice, inhibition of DNA-PK significantly attenuated neointimal lesion size 3 weeks after wire-injury. Conclusion DNA-PK directly phosphorylates NOR-1 and, this way, modulates SMC proliferation. These data add to our understanding of vascular remodelling processes and opens new avenues for treatment of vascular proliferative diseases.

BACKGROUND: The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN's effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury in vivo and in vitro. METHODS AND RESULTS: Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H2O2 robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; P

Dedifferentiation, migration, and proliferation of resident vascular smooth muscle cells (SMCs) are key components of neointima formation after vascular injury. Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in this process, but the complex regulation of STAT3-dependent genes and the functional significance of inhibiting this pathway during the development of vascular proliferative diseases remain elusive. In this study, we demonstrate that STAT3 was activated in neointimal lesions following wire-induced injury in mice. Phosphorylation of STAT3 induced trans-activation of cyclin D1 and survivin in SMCs in vitro and in neointimal cells in vivo, thus promoting proliferation and migration of SMCs as well as reducing apoptotic cell death. WP1066, a highly potent inhibitor of STAT3 signaling, abrogated phosphorylation of STAT3 and dose-dependently inhibited the functional effects of activated STAT3 in stimulated SMCs. The local application of WP1066 via a thermosensitive pluronic F-127 gel around the dilated arteries significantly inhibited proliferation of neointimal cells and decreased the neointimal lesion size at 3 weeks after injury. Even though WP1066 application attenuated the injury-induced up-regulation of the chemokine RANTES at 6 h after injury, there was no significant effect on the accumulation of circulating cells at 1 week after injury. In conclusion, these data identify STAT3 as a key molecule for the proliferative response of SMC and neointima formation. Moreover, inhibition of STAT3 by the potent and specific compound WP1066 might represent a novel and attractive approach for the local treatment of vascular proliferative diseases. Electronic supplementary material The online version of this article (doi:10.1007/s00395-012-0261-9) contains supplementary material, which is available to authorized users.

The proliferation and migration of vascular smooth muscle cells (SMCs) from the media toward the intimal layer are key components in vascular proliferative diseases. In addition, the differentiation of circulating bone marrow-derived mononuclear cells (BMMCs) into SMCs has been described to contribute to lesion progression in experimental models of atherosclerosis, transplant arteriosclerosis, and neointima formation. In vitro, CD14(+) BMMCs from peripheral blood acquire a spindle-shaped phenotype and express specific SMC markers in response to platelet-derived growth factor-BB. However, the 'trans-differentiation' capacity of BMMCs into definitive SMCs in vivo remains a highly controversial issue. Whereas SMCs within atherosclerotic plaques have been demonstrated to be exclusively of local origin, more severe injury models have shown a wide diversity of SMCs or smooth muscle-like cells derived from BMMCs. In hindsight, these discrepancies may be attributed to methodological differences, e.g., the use of high-resolution microscopy or the specificity of the SMC marker proteins. In fact, the analysis of mouse strains that express marker genes under the control of a highly specific smooth muscle-myosin heavy chain (SM-MHC) promoter and a time-course analysis on the dynamic process of neointima formation have recently shown that BMMCs temporarily express α-smooth muscle actin, not SM-MHC. Additionally, BM-derived cells disappear from the neointimal lesion after the inflammatory response to the injury has subsided. Although CD14(+)/CD68(+) have important paracrine effects on arterial lesion progression, BMMCs account for more of the 'SMC-like macrophages' than the highly 'trans-differentiated' and definitive SMCs in vivo. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidyl inositol-anchored protein that mediates cell adhesion to the extracellular matrix protein vitronectin (VN). We demonstrate here that this cell adhesion process is accompanied by the formation of an adhesion patch characterized by an accumulation of uPAR into areas of direct contact between the cell and the matrix. The adhesion patch requires the glycolipid anchor and develops only on a VN-coated substrate, but not on fibronectin. It consists of detergent-insoluble microdomains that accumulate F-actin and tyrosine-phosphorylated proteins, but not beta(1) integrins. Lack of inhibition of adhesion in the presence of integrin-blocking reagents and adhesion on a VN fragment without the RGD sequence indicated that the adhesion of uPAR-bearing cells on VN could occur independently of integrins. Hence, uPAR-mediated cell adhesion on VN relies on the formation of a unique cellular structure that we have termed "detergent-insoluble adhesion patch" (DIAP).