Intermittent Fasting After ST-Segment-Elevation Myocardial Infarction Improves Left Ventricular Function: The Randomized Controlled INTERFAST-MI Trial.

BACKGROUND: Intermittent fasting has shown positive effects on numerous cardiovascular risk factors. The INTERFAST-MI trial (Intermittent Fasting in Myocardial Infarction) has been designed to study the effects of intermittent fasting on cardiac function after STEM (ST-segment-elevation myocardial infarction) and the feasibility of future multicenter trials. METHODS: The INTERFAST-MI study was a prospective, randomized, controlled, nonblinded, single-center investigator-initiated trial. From October 1, 2020, to July 15, 2022, 48 patients were randomized to the study groups intermittent fasting or regular diet and followed for 6 months with follow-up visits at 4 weeks and 3 months. RESULTS: In all, 22 of 24 patients in the intermittent fasting group with a mean age of 58.54±12.29 years and 20 of 24 patients in the regular diet group with a mean age of 59.60±13.11 years were included in the intention-to-treat population. The primary efficacy end point (improvement in left ventricular ejection fraction after 4 weeks) was significantly greater in the intermittent fasting group compared with the control group (mean±SD, 6.636±7.122%. versus 1.450±4.828%; P=0.038). This effect was still significant and even more pronounced after 3 and 6 months. The patients in the intermittent fasting group showed a greater reduction in diastolic blood pressure and body weight compared with the control group. The mean adherence of patients in the intermittent fasting group was a median of 83.7% (interquartile range, 69.0%-98.4%) of all days. None of the patients from either group reported dizziness, syncope, or collapse. CONCLUSIONS: Our results suggest that intermittent fasting after myocardial infarction may be safe and could improve left ventricular function after STEMI. REGISTRATION: URL: https://www.drks.de; Unique identifier: DRKS00021784.

Isolation of circulating endothelial cells provides tool to determine endothelial cell senescence in blood samples.

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.

Empagliflozin prevents neointima formation by impairing smooth muscle cell proliferation and accelerating endothelial regeneration.

Background: Empagliflozin, an inhibitor of the sodium glucose co-transporter 2 (SGLT2) and developed as an anti-diabetic agent exerts additional beneficial effects on heart failure outcomes. However, the effect of empagliflozin on vascular cell function and vascular remodeling processes remains largely elusive. Methods/Results: Immunocytochemistry and immunoblotting revealed SGLT2 to be expressed in human smooth muscle (SMC) and endothelial cells (EC) as well as in murine femoral arteries. In vitro, empagliflozin reduced serum-induced proliferation and migration of human diabetic and non-diabetic SMCs in a dose-dependent manner. In contrast, empagliflozin significantly increased the cell count and migration capacity of human diabetic ECs, but not of human non-diabetic ECs. In vivo, application of empagliflozin resulted in a reduced number of proliferating neointimal cells in response to femoral artery wire-injury in C57BL/6J mice and prevented neointima formation. Comparable effects were observed in a streptozocin-induced diabetic model of apolipoprotein E-/- mice. Conclusive to the in vitro-results, re-endothelialization was not significantly affected in C57BL/6 mice, but improved in diabetic mice after treatment with empagliflozin assessed by Evan's Blue staining 3 days after electric denudation of the carotid artery. Ribonucleic acid (RNA) sequencing (RNA-seq) of human SMCs identified the vasoactive peptide apelin to be decisively regulated in response to empagliflozin treatment. Recombinant apelin mimicked the in vitro-effects of empagliflozin in ECs and SMCs. Conclusion: Empagliflozin significantly reduces serum-induced proliferation and migration of SMCs in vitro and prevents neointima formation in vivo, while augmenting EC proliferation in vitro and re-endothelialization in vivo after vascular injury. These data document the functional impact of empagliflozin on vascular human SMCs and ECs and vascular remodeling in mice for the first time.

Effect of intermittent fasting after ST-elevation myocardial infarction on left ventricular function: study protocol of a pilot randomised controlled trial (INTERFAST-MI).

INTRODUCTION: Preclinical studies consistently show robust disease-modifying effects of intermittent fasting in animal models of cardiovascular disease. However, the impact of intermittent fasting on cardiovascular endpoints after myocardial infarction has not been investigated in a clinical trial so far. METHODS AND ANALYSIS: The INTERmittent FASTing after Myocardial Infarction (INTERFAST-MI) trial is a monocentric prospective randomised controlled non-confirmatory pilot study including 48 patients with ST-segment elevation myocardial infarction. They will be randomised in a 1:1 ratio to either intermittent fasting (daily time-restricted eating; consuming food for not more than 8 hours/day, fasting for at least 16 hours/day) or to a control group without a particular diet. The follow-up time is 6 months. The prespecified primary outcome is change in left ventricular systolic function at 4 weeks from baseline to estimate effect size required to establishing sample size and power calculation for a future full-scale trial. Secondary outcomes include protocol adherence, recruitment, major adverse cardiac events, revascularisation, changes in left ventricular systolic function at 3 and 6 months, patient weight, blood pressure, and serum markers of inflammation and cardiovascular disease. Enrolment began on 1 November 2020 and is expected to conclude in December 2021. ETHICS AND DISSEMINATION: The trial has received ethics approval from the Medical Ethics Committee of the Martin-Luther-University Halle-Wittenberg. Results of the study will be submitted for publication in a peer-reviewed journal and presented at scientific conferences. TRIAL REGISTRATION NUMBER: DRKS00021784.

BET bromodomain-containing epigenetic reader proteins regulate vascular smooth muscle cell proliferation and neointima formation.

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.

The differential statin effect on cytokine production of monocytes or macrophages is mediated by differential geranylgeranylation-dependent Rac1 activation.

Monocytes and macrophages contribute to pathogenesis of various inflammatory diseases, including auto-inflammatory diseases, cancer, sepsis, or atherosclerosis. They do so by production of cytokines, the central regulators of inflammation. Isoprenylation of small G-proteins is involved in regulation of production of some cytokines. Statins possibly affect isoprenylation-dependent cytokine production of monocytes and macrophages differentially. Thus, we compared statin-dependent cytokine production of lipopolysaccharide (LPS)-stimulated freshly isolated human monocytes and macrophages derived from monocytes by overnight differentiation. Stimulated monocytes readily produced tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Statins did not alter cytokine production of LPS-stimulated monocytes. In contrast, monocyte-derived macrophages prepared in the absence of statin lost the capacity to produce cytokines, whereas macrophages prepared in the presence of statin still produced cytokines. The cells expressed indistinguishable nuclear factor-kB activity, suggesting involvement of separate, statin-dependent regulation pathways. The presence of statin was necessary during the differentiation phase of the macrophages, indicating that retainment-of-function rather than costimulation was involved. Reconstitution with mevalonic acid, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate blocked the retainment effect, whereas reconstitution of cholesterol synthesis by squalene did not. Inhibition of geranylgeranylation by GGTI-298, but not inhibition of farnesylation or cholesterol synthesis, mimicked the retainment effect of the statin. Inhibition of Rac1 activation by the Rac1/TIAM1-inhibitor NSC23766 or by Rac1-siRNA (small interfering RNA) blocked the retainment effect. Consistent with this finding, macrophages differentiated in the presence of statin expressed enhanced Rac1-GTP-levels. In line with the above hypothesis that monocytes and macrophages are differentially regulated by statins, the CD14/CD16-, merTK-, CX3CR1-, or CD163-expression (M2-macrophage-related) correlated inversely to the cytokine production. Thus, monocytes and macrophages display differential Rac1-geranylgeranylation-dependent functional capacities, that is, statins sway monocytes and macrophages differentially.

Targeting of Extracellular RNA Reduces Edema Formation and Infarct Size and Improves Survival After Myocardial Infarction in Mice.

BACKGROUND: Following myocardial infarction (MI), peri-infarct myocardial edema formation further impairs cardiac function. Extracellular RNA (eRNA) released from injured cells strongly increases vascular permeability. This study aimed to assess the role of eRNA in MI-induced cardiac edema formation, infarct size, cardiac function, and survival after acute MI and to evaluate the therapeutic potential of ribonuclease 1 (RNase-1) treatment as an eRNA-degrading intervention. METHODS AND RESULTS: C57BL/6J mice were subjected to MI by permanent ligation of the left anterior descending coronary artery. Plasma eRNA levels were significantly increased compared with those in controls starting from 30 minutes after ligation. Systemic application of RNase-1, but not DNase, significantly reduced myocardial edema formation 24 hours after ligation compared with controls. Consequently, eRNA degradation by RNase-1 significantly improved the perfusion of collateral arteries in the border zone of the infarcted myocardium 24 hours after ligation of the left anterior descending coronary artery, as detected by micro-computed tomography imaging. Although there was no significant difference in the area at risk, the area of vital myocardium was markedly larger in mice treated with RNase-1 compared with controls, as detected by Evans blue and 2,3,5-triphenyltetrazolium chloride staining. The increase in viable myocardium was associated with significantly preserved left ventricular function, as assessed by echocardiography. Moreover, RNase-1 significantly improved 8-week survival following MI. CONCLUSIONS: eRNA is an unrecognized permeability factor in vivo, associated with myocardial edema formation after acute MI. RNase-1 counteracts eRNA-induced edema formation and preserves perfusion of the infarction border zone, reducing infarct size and protecting cardiac function after MI.