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. 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. 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.

Circulating endothelial cells (CEC) are arising as biomarkers for vascular diseases. However, whether they can be utilized as markers of endothelial cell (EC) senescence in vivo remains unknown. Here, we present a protocol to isolate circulating endothelial cells for a characterization of their senescent signature. Further, we characterize different models of EC senescence induction in vitro and show similar patterns of senescence being upregulated in CECs of aged patients as compared to young volunteers. Replication-(ageing), etoposide-(DNA damage) and angiotensin II-(ROS) induced senescence models showed the expected cell morphology and proliferation-reduction effects. Expression of senescence-associated secretory phenotype markers was specifically upregulated in replication-induced EC senescence. All models showed reduced telomere lengths and induction of the INK4a/ARF locus. Additional p14ARF-p21 pathway activation was observed in replication- and etoposide-induced EC senescence. Next, we established a combined magnetic activated- and fluorescence activated cell sorting (MACS-FACS) based protocol for CEC isolation. Interestingly, CECs isolated from aged volunteers showed similar senescence marker patterns as replication- and etoposide-induced senescence models. Here, we provide first proof of senescence in human blood derived circulating endothelial cells. These results hint towards an exciting future of using CECs as mirror cells for in vivo endothelial cell senescence, of particular interest in the context of endothelial dysfunction and cardiovascular diseases.

(1) Background: Hemoadsorption is a method of blood purification with a wide spectrum of indications. Pre-emptive use of hemoadsorption in patients undergoing heart surgery with cardiopulmonary bypass is considered to reduce the risk of postoperative systemic inflammatory response syndrome. The current study aimed to identify the spectrum of blood proteins adsorbed on the polymer matrix of the CytoSorb hemoadsorption system and to investigate their influence on cultured endothelial cells in vitro. (2) Methods: Adsorbers used for intraoperative hemoadsorption were obtained from patients undergoing on-pump valve surgery in acute endocarditis. Proteins were extracted from the adsorbers, purified, identified with mass-spectrometry and applied to cultured human aortic endothelial cells. (3) Results: A broad range of blood proteins were identified in the material eluted from the CytoSorb adsorber. When added to cultured ECs, these protein extracts caused severe reduction in cell viability and migration. After 24 h exposure, transcriptional changes with up-regulation of multiple metabolic regulators were observed and verified on the protein level. Genes responsible for control of mitosis were significantly down-regulated. (4) Conclusions: In summary, our data reveal that intraoperative hemoadsorption allows broad spectrum removal of a wide range of molecules eliciting endothelial damage.

Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases and are characterized by a reduced angiogenic and regenerative potential. Therefore, targeting these cells has been suggested as an effective therapeutic strategy to reduce vascular disease burden and potentially improve health and lifespan of humans. Here, we aimed to establish a pharmacological, partial reprogramming strategy to improve replicative senescent endothelial cell function in the context of angiogenesis. We demonstrate that our treatment improves tube formation and sprouting capacity but also increases proliferation and migration capacity in vitro. Further, inflammation and DNA damage were reduced in the replicative senescent cells. These processes were initiated by a short and timely-restricted overexpression of the Yamanaka-factors induced by our pharmacological strategy. The advantage of these compounds is that they are FDA approved in their respective concentrations which could pave the way for use in a clinical setting.

Background: Vascular restenosis resulting from neointima formation significantly limits the efficacy of percutaneous interventional therapies compared with bypass surgery. The adventitial layer is involved in neointima formation, but the detailed pathophysiological interplay of the different cell types in this process is still unclear. Methods: We analyzed the correlation between adventitial and neointimal tissue size in human postmortem restenotic lesions after angioplasty. In porcine and mouse models of vascular injury, we examined early proliferation of fibroblasts and adventitial expansion. Using anti‐CD45 antibodies, we identified recruited leukocytes as the source of fibroblast activation following vascular injury in mice. A time‐course experiment on neointima formation demonstrated that adventitial activation precedes the proliferation of medial and neointimal smooth muscle cells (SMCs). To further investigate this process, we developed a mouse model enabling the surgical removal and transplantation of adventitial tissue. Results: We observed that activated adventitial fibroblasts release interleukin 6 and other cytokines, which strongly induce SMC proliferation and migration in vitro. In interleukin 6 knockout mice, supernatants from activated adventitia grafts failed to stimulate SMC proliferation and migration. Furthermore, transplantation of adventitial grafts from interleukin 6 knockout mice did not induce neointima formation. Cell fate tracking experiments using double transgenic reporter mice demonstrated that resident adventitial cells do not directly contribute to the neointimal cellular mass. Instead, medial SMCs were identified as the primary source of neointimal cells. Conclusions: We show that the release of interleukin 6 by adventitial fibroblasts induces the subsequent proliferation and migration of medial SMC in the process of neointima formation. Thus, we propose a new paradigm for adventitial fibroblasts in this process as a paracrine inflammatory engine. Anti‐inflammatory targeting of the vascular adventitia might thus be promising to limit neointima formation.

Vascular aging, endothelial dysfunction, and cellular senescence are interconnected processes that drive cardiovascular diseases (CVDs). Several studies have confirmed that microRNAs (miRNAs) are closely associated with the development of CVDs. Among them, miR-21-5p and miR-146a-5p have emerged as critical players in CVDs: MiR-21-5p is implicated in promoting inflammation and fibrosis, while miR-146a-5p influences vascular smooth muscle cell proliferation and inflammatory responses. Targeting these miRNAs for therapeutic intervention could provide a novel strategy to mitigate the deleterious effects of vascular aging. Recent advances in RNA-based therapeutics have led to the exploration of circular RNAs (circRNAs) as stable and effective miRNA inhibitors. Due to their circular structure, circRNAs exhibit greater stability than linear miRNA inhibitors, such as anti-miRs. This study aimed to evaluate the expression of miR-21-5p and miR-146a-5p in senescent human vascular endothelial cells and vascular smooth muscle cells (VSMCs) and to assess whether synthetic circRNAs could effectively inhibit these miRNAs. In a series of experiments, human umbilical vein endothelial cells (HUVECs) and VSMCs were transfected with custom-designed circRNAs targeting these miRNAs. Here, we want to focus on VSMCs. The effects on miRNA expression, target gene regulation, and cellular functions such as proliferation and migration were analyzed. Our findings demonstrate that circRNA-mediated inhibition of miR-21-5p and miR-146a-5p significantly affects gene expression and cellular behavior in senescent vascular cells, suggesting that circRNAs may serve as more efficient and stable therapeutic agents compared to traditional anti-miRs. By addressing these objectives, this study contributes to the growing body of knowledge on circRNA-mediated miRNA inhibition and its potential implications for vascular health. The results from this study not only enhance our understanding of miR-21-5p and miR-146a-5p in vascular senescence but also provide a foundation for future therapeutic strategies.