Myocardial infarction (MI) is a common cardiovascular disease caused by permanent loss of cardiomyocytes and the formation of scar tissue due to myocardial ischemia. Mammalian cardiomyocytes lose their ability to proliferate almost completely in adulthood and are unable to repair the damage caused by MI. Therefore, transplantation of exogenous cells into the injured area for treatment becomes a promising strategy. Pluripotent stem cells (PSCs) have the ability to proliferate and differentiate into various cellular populations indefinitely, and pluripotent stem cell-derived cardiomyocytes (PSC-CMs) transplanted into areas of injury can compensate for part of the injuries and are considered to be one of the most promising sources for cell replacement therapy. However, the low transplantation rate and survival rate of currently transplanted PSC-CMs limit their ability to treat MI. This article focuses on the strategies of current research for improving the therapeutic efficacy of PSC-CMs, aiming to provide some inspiration and ideas for subsequent researchers to further enhance the transplantation rate and survival rate of PSC-CMs and ultimately improve cardiac function. Myocardial infarction (MI), also called heart attack, is the most common form of cardiovascular disease and a leading cause of mortality worldwide . MI invode novo cardiomyocytes. Among the cell types currently used for therapeutic purposes are pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); adult stem cells ; and somatic cells   . While aoblasts) .While ESCs are derived from the inner cell mass of the embryonic blastocyst, iPSCs are similar to ESCs and are generated from somatic cells by the transduction of different reprogramming factors, e.g., c-MYC, Klf4, Oct4, and Sox2. There are potential challenges with the use of iPSCs and ESCs in cell therapy that must be addressed to minimize the drawbacks and enhance the benefits. Transplanted exogenous ESCs often elicit immune rejection, which markedly diminishes the therapeutic effect or causes further damage; immune rejection can be suppressed by HLA matching and knocking down the antigen-encoding gene. In contrast, iPSCs derived from autologous cells do not evoke severe immune rejection by the organism . The PSCFaced with the limitations of PSC-CM therapies for MI, one possible strategy is to pretreat PSC-CMs before transplantation. Many drugs have been shown to be efficacious in MI, which has led investigators to wonder whether the pretreatment of PSC-CMs with these drugs could improve their efficacy in the treatment of MI. Additionally, pretreatment with non-drug factors, such as hypoxia and starvation, has also been shown to somewhat alleviate the limitations of PSC-CM therapy.in vivo study in a mouse model found that transplantation of human iPSC-CMs into myocardial infarct areas after combination pretreatment with CHIR99021 and FGF1 increased cell cycle activity by approximately 4–6-fold and the transplantation rate by approximately 4-fold. The effect of CHIR99021 or FGF1 pretreatment alone was weaker than that of cotreatment but significantly stronger than that of the control  may be promising. The fibroblast growth factor (FGF) family, also known as the heparin affinity growth factor family, is a class of peptide-like molecules that bind to specific cell membrane receptors. Fibroblast growth factor 1 (FGF1) is involved in several physiological processes, such as development, morphogenesis and proliferation, and FGF1 treatment for acute myocardial injury was found to increase cardiomyocyte mitosis, reduce scar area, and improve cardiac function . FGF21 a control .in vitro that Tb4 could increase AKT activity and BcL-XL expression to protect cultured hiPSC-CMs from hypoxic injury. In a porcine MI model, the combined effect of human iPSC-CMs and Tb4 improved the engraftment rate, vascularization and cardiac function, and no tumor tissue was found in this immunodeficient porcine model  was found to promote cardiomyocyte survival and proliferation and to enhance angiogenesis. Tan et al. demonstrated ne model . The enhne model . Y-276322+ channel that can be activated by binding to DAG and mediates Ca2+ influx, indicating that TRPC7 is an important potential drug target  channels play an important role in regulating the automaticity of ESC-CMs. TRPC7 is a receptor-operated Cag target . Similarg target , and thein vivo and found that EBSS-induced starvation significantly promoted the structural, metabolic and electrophysiological maturation of PSC-CMs. Moreover, this treatment increased the mitochondrial content of ESC-CMs and enhanced their oxidative phosphorylation capacity  to simulate starvation capacity . ESCs arcapacity . Hu et acapacity . TherefoAdult mammalian cardiomyocytes have almost no proliferative capacity; thus, it is difficult to replenish new cardiomyocytes after injury, an important reason for the dramatic impairment of cardiac function after MI. In contrast, during mammalian fetal development, cardiomyocytes have the ability to proliferate, but soon after birth, their proliferative capacity begins to decline until it is lost. This loss of proliferative ability mainly stems from the downregulation of cell cycle maintenance factors as the heart matures, which causes cardiomyocytes to enter G0 of the cell cycle and thus fail to provide new cells at the site of injury after MI. Therefore, researchers have explored ways to increase the proliferative capacity of PSC-CMs from the perspective of gene expression regulation. Overexpressing cell cycle maintenance factors in PSC-CMs may prolong cell cycle progression, allowing more cardiomyocytes to remain in the proliferative phase and increasing the retention of transplanted cells in the infarct zone. Cell cycle protein D (cyclin D) regulates the cell cycle at the G1/S transition, wherein it binds to Cyclin Dependent Kinase 1 (CDK1) and activates CDK1 activity through a series of phosphorylation and dephosphorylation events to move cells from G1 to the replicative S phase. Zhu et al. showed that the overexpression of cyclin D2 in human iPSC-CMs increased graft size and improved myocardial recovery in a mouse model of MI by increasing the proliferation of transplanted cells, which played a significant role in myocardial remyelination and ventricular function recovery . Zhao etAn increased number of PSC-CMs lingering in the infarct zone and increased cell cycle activity can also be achieved by reducing cell death. Many genes are closely related to cell aging and apoptosis, and regulating the expression of these genes can slow cell aging and inhibit apoptosis. Engel et al. showed that p38 activity is one important component of cell cycle regulation in the mammalian myocardium. p38 activity is negatively correlated with cardiac growth, and p38 overexpression inhibits myocardial proliferation . p38 belIn addition to increasing the number of PSC-CMs in the MI region by promoting cell proliferation and reducing apoptosis, enhancing the integration between transplanted PSC-CMs and host cells can also improve the therapeutic effect on MI. By expressing certain exogenous genes in PSC-CMs, graft vascularization and cellular junctions between donor and host cells can be improved to achieve functional integration. Tao et al. transfected Ang-1 into human iPSC-CMs and then implanted these genetically altered cells into the anterior wall of the left ventricle after MI in rats. The results showed that Ang-1 improved the survival rate of iPSC-CMs after transplantation, induced mitosis and small artery formation in both host and donor cells, and improved the cell engraftment rate . Lou et K1) channels in a variety of cell types, including cardiomyocytes; therefore, Liao et al. overexpressed the ion channel Kir2.1 in ESC-CMs. IK1 can hyperpolarize the resting membrane potential, and IK1 expression seems to be a marker of ESC-CM maturation. Although ESC-CMs improved cardiac function after MI with or without the overexpression of Kir2.1, the risk of ventricular tachyarrhythmias and sudden death was significantly reduced by Kir2.1 overexpression , fibroblasts, and endothelial cells (ECs). Therefore, one idea for improving the therapeutic effect after MI is to transplant PSC-CMs into the infarcted area together with other cells or tissues that could ensure a high survival rate of the transplanted PSC-CMs through paracrine signaling and provide a suitable microenvironment for PSC-CMs. This approach has been shown to significantly improve the therapeutic efficacy of PSC-CMs in MI. Sekine et al. cocultured ECs with cardiomyocytes from newborn mice, and these ECs formed a network structure in cardiomyocyte sheets that significantly increased vascular structure and cytokine secretion; in addition, engineered myocardial tissue consisting of three layers of cardiomyocyte sheets was transplanted into a MI model and significantly improved cardiac function . This fiCotransplantation of PSC-CMs with other cells or tissues improves cardiac function through two main ways: promoting vascularization and increasing the secretion of paracrine factors. Vascular regeneration of the graft in the infarct area can provide oxygen and nutrients to the transplanted cells and improve the survival rate of transplanted PSC-CMs. Moreover, the graft provides a favorable microenvironment that promotes cardiomyocyte proliferation, survival, and vascularization by prolonging the secretion of paracrine factors. Park et al. used a patch of human MSCs in conjunction with iPSC-CMs to promote myocardial repair . MSCs sein vitro experiments showed that exosomes protect iPSC-CMs and promote vascularization by inhibiting apoptosis, maintaining intracellular calcium homeostasis, and increasing adenosine 5′-triphosphate synthesis  using PSC-derived cardiovascular cells in a ratio of 15% endothelial cells, 70% cardiomyocytes, and 15% fibroblasts, showing that PSC-CMs in MTs have improved sarcomere structures with T-tubules, increased contractility, and more mature electrophysiological phenotypes than PSC-CMs alone . The maiMyocardial regeneration strategies have been hampered by the lack of cardiac cell sources and the marked loss of donor cells after transplantation. PSC-CMs are a potential adequate cell source for cell replacement therapy but do not solve the problem of rapid donor cell loss. Cardiac tissue engineering is an interdisciplinary strategy in which bionic scaffolds and seed cells are used to generate tissue replacements aimed at restoring the structure, morphology, and function of damaged cardiac tissue . CommonlThe developmental maturation of cardiomyocytes requires interactions among various regulatory mechanisms within the cell, but the extracellular matrix also plays an important role . Samura in vivo myocardial environment and promote the maturation of PSC-CMs, with better myocardial myofibrillar alignment and more ordered electrical properties , and then transplanted induced vascular progenitor cells (iVPC) together with 3D cell/polymer micro-beams formed by micro-scaffolds into a mouse model of myocardial infarction, and the results showed that iVPC on microbeam scaffolds had higher survival rates and the ability to treat infarction . SimilarPSC-CMs can provide a suitable cell source for cell replacement therapy. Transplanting PSC-CMs into the heart can restore a portion of the cell loss and scar tissue caused by MI, thus restoring normal physiological function of the heart . A majorTherefore, to address these problems, scientists have used strategies such as PSC-CM pretreatment, gene expression modulation, cotransplantation with other cells and cardiac tissue engineering to mitigate the limitations of PSC-CM therapies and increase their efficacy in the treatment of MI. Other investigators have developed combination strategies that can achieve better results. Kawamura et al. found that combining cell sheets with the Omental Flap Technique enhanced the therapeutic effect of human iPSC-CMs in a porcine ischemic cardiomyopathy model . AlternaWith regard to the clinical translation of autologous stem cell therapy in acute myocardial infarction, two cell types have now completed phase III clinical trials: skeletal myogenic cells and bone marrow mononuclear cells. Large-scale randomized clinical studies have also demonstrated the clinical feasibility and safety of transcoronary infusion of autologous bone marrow-derived mononuclear cells and circulating progenitor cells in patients with acute MI, showing a 9.3% increase in left ventricular ejection fraction and a reduction in myocardial infarct size at 12 months follow-up . Ideallyin vitro experiments; most studies have used mouse, rat, and porcine models of MI, with few performed in primates. The clinical relevance of animal models is limited by significant physiological differences in size, electrical conduction, and coronary artery formation and distribution between the animal and human heart. Therefore, rigorous clinical trials and the establishment of standard operating procedures are needed to address the safety issues associated with PSC-CM transplantation, such as whether this procedure will cause new heart diseases or lead to tumorigenesis. In addition, the ethical issues associated with the transplantation of PSC-CMs are a major impediment to clinical use. Meanwhile, a standardized and scaled-up culture protocol for PSC-CM treatment in clinical settings is still lacking, and further collaborative research by researchers and medical professionals is needed. Future issues to be addressed in clinical trials may also include determining the appropriate timing of transplantation and the number of cells transplanted. The appropriate number of cells to be transplanted must be determined to ensure efficacy without causing cardiac arrhythmias due to excessive cell numbers. In the future, these strategies can be combined with conventional pharmacological and surgical treatments , which may further improve the therapeutic efficacy of PSC-CMs.Current research on PSC-CM replacement therapy is limited to animal and When performing cell transplantation, some undifferentiated and partially differentiated PSCs might be also transplanted together with PSC-CMs into the infarcted region. Since undifferentiated PSCs exhibit intrinsic tumorigenic properties that allow them to form teratomas, cell therapy products containing residual undifferentiated PSCs may lead to tumor formation after transplantation. It has been shown that transplantation of different iPSC lines shows significant differences in tumor incidence, latency of formation, and volume. Cho et al. developed a PSC-specific fluorescent probe, CDy1, which generates reactive oxygen species (ROS) upon visible light irradiation that induces selective PSCs death. Importantly, CDy1 and light irradiation did not have a negative effect on differentiated endothelial cells. Photodynamic treatment of PSCs with CDy1 and visible light irradiation confirmed the inhibition of teratoma formation in mice . StudiesAlthough PSC-CMs still predominantly act in a paracrine manner due to low transplantation efficiency and many issues remain to be resolved to achieve clinical translation, the great potential for the treatment of MI encourages researchers to continue exploring and developing better protocols. We believe that with the optimization of PSC-CM pretreatment and transplantation techniques, the goal of PSC-CM transplantation to repair the necrotic myocardium will eventually be achieved.


Background: Most of the drugs approved and registered for use in heart failure (HF) therapy were examined in randomized clinical trials (RCTs) with the primary composite endpoint of death or hospital readmission. This study aimed to analyze the rates of the newly calculated event: death without prior hospital readmission, in HFrEF patients in large RCTs to show that the newly defined endpoint probably delivers additional data on the structure of the composite endpoint and helps to interpret the results of interventional studies. Methods: This study included RCTs on therapeutic interventions in HF patients. A literature search was performed, and 31 trials in which death without hospital admission could be calculated were included in the analyses. The death without a prior hospital admission endpoint was calculated as the difference between the composite endpoint rate  and the readmission rate. The differences in the new endpoint between the study groups were calculated. Result: The death rates without prior hospital admission were lower in the intervention groups in five trials. In the SENIORS study, significant differences were found in the primary (composite) and death without previous hospital admission endpoints. In the ACCLAIM, VEST, and GISSI-HF STATIN trials, death without previous hospital admission was the only endpoint with a significant difference between the study groups. Moreover, the new endpoint rates were higher in the intervention group in the latter two studies. Conclusions: The new endpoint describing patients who died without prior hospital admission might be useful in previous and future interventional studies to provide additional data on the structure of the composite endpoint. Some therapies might reduce death without previous hospital admission rates, which could be beneficial, even without a reduction in overall long-term mortality. Poor outcomes characterize heart failure with reduced ejection fraction (HFrEF). The one-year all-cause mortality may exceed 20% while rates of one-year hospital readmission for heart failure and any cause reach 17% and 48%, respectively . It was This study included RCTs on drugs, non-drug interventions, and device therapies in patients with HFrEF. As some trials were designed before the current definition of HFrEF, the inclusion criteria regarding the left ventricular ejection fraction (LVEF) were not the same. Thus, all trials with decreased LVEF, regardless of the cut-off threshold, were included.A literature search was performed in September and October 2020 to select RCTs fulfilling the eligibility criteria. Two authors searched the MEDLINE database independently to screen titles and abstracts using the predefined protocol with the search query:“(heart failure [Title]) AND ((death [Title/Abstract]) or  or (composite endpoint [Title/Abstract]))”.The search results included 19,157 studies evaluated according to the flowchart .This study included RCTs that enrolled patients with HF/HF with decreased EF with drug or non-drug intervention or device therapy with the following endpoints: composite endpoint consisting of death or hospital admission and hospital admission; cause of hospital admission had to be the same as in the composite endpoint .The analysis excluded trials with follow-up shorter than 30 days or trials discontinued early or suspended or with more than two parameters in the composite endpoint , or trials enrolling patients with asymptomatic left ventricular dysfunction instead of HF.All disagreements were referred to as the third researcher, who made a final decision (and did not fulfill the criteria for being an author).The list of included HF trials contains the trial name, study drug/device/intervention, year of publication, enrolment dates, number of participants, follow-up, cause of death, and cause of hospital admissionThe characteristics of patients included in the analyzed HF trials, including age, sex, HF etiology, HF duration, percent NYHA III/IV class, LVEF, heart rate, percent of implantable cardioverter-defibrillators (ICDs), cardiac resynchronization therapy with defibrillator (CRT-D), diuretics, beta-blockers, angiotensin-converting enzyme inhibitor (ACEI)/angiotensin receptor blockers (ARBs)/angiotensin receptor-neprilysin inhibitor (ARNI), and mineralocorticoid receptor antagonist (MRA) use.Death from CV causes without hospital admission for HF/worsening HF;Death for any reason without hospital admission;Death for any reason without CV hospital admission;Death for any reason without CV hospital admission;Death for any reason without any hospital admission;Death for CV reason without any hospital admission;Death for HF worsening without any hospital admission for HF worsening.Tables with different endpoints’ combinations with trial name, study groups, follow-up, composite endpoint rates, death, and hospital admission. Based on the extracted data on events: death or hospitalization and death alone, the new endpoint—death without rehospitalization—was calculated as the difference between the two events . DependiThe full texts of all selected articles were obtained. The data was extracted to the five predefined templates: As no raw data is available regarding the mortality and follow-up (time to death), the difference between the death without hospital readmission rates was calculated using a 2 × 2 chi-square test . The analysis included 31 trials, of which 18 were progressive-positive and 13 progressive-negative trials presented in The detailed rates of events are presented in The number of trials and death rates without previous hospital admission, depending on their cause, are presented in The detailed data on the endpoint rates in trials with a significant difference between groups regarding the death without hospital admission endpoint is presented in Death without previous hospital admission is an easy to calculate endpoint in clinical trials as a difference between the rate of a composite endpoint of death or hospital admission and the rehospitalization rate. Depending on the cause of death and hospital admission in the composite endpoint, death rates without previous hospital admission may have different clinical interpretations and significance . For exaSeven studies showed significant differences between the trial groups regarding death rates without prior hospital admission. In the PARADIGM-HF trial, a reduction in CV death without prior HF hospitalization was observed. Assuming that all acute HF cases were hospitalized, it may suggest that the trial drug reduced the number of CV deaths due to non-HF reasons or reduced the number of sudden cardiac deaths. In the MERIT-HF study, the intervention reduced all-cause death without prior HF hospitalization, suggesting the study drug’s positive impact on deaths other than HF-caused deaths. In EPHESUS and SENIOR trials, reduced CV mortality without previous CV hospitalization was achieved, which may describe the sudden cardiac death or death in the terminal phase of the disease when hospitalization might be intentionally avoided. Patients with CV diseases and a substantial risk of CV death are supposed to be hospitalized before death. In the SENIORS trial, the rates of CV death and separate hospital admission due to CV reasons did not differ between groups, and the only significant differences were found in the primary (composite) and death without previous hospital admission endpoints. Thus, the whole trial’s effect was probably driven by the differences in death rates without prior hospital admission.In the ACCLAIM trial, death without previous hospital admission was the only endpoint with a significant difference between the study groups, as immune therapy reduced mortality without previous hospital admission with no effect on conventional endpoints. Using the new endpoint, the trial conclusions could influence future scientific directions regarding immune therapy in HF and possibly clinical recommendations. In another two trials without positive results in HF patients (VEST and GISSI-HF STATIN), death without previous hospital admission was the only endpoint with a significant difference between the study groups. Moreover, in both studies, the death rates without previous hospital admission were higher in the intervention group, meaning that patients in the placebo groups died more often in the hospital than in the intervention groups. It might be indirect proof of higher rates of sudden cardiac death in the intervention groups, as there is no reason for higher hospital mortality rates in patients in the placebo groups. It could also explain why the primary trial endpoints did not reach statistical significance .Deaths without previous HF hospitalization may be a valuable clinical outcome and RCT event. However, contrary to hospital admissions due to myocardial infarction, not all HF hospitalizations are urgent. Hospital admissions due to HF may include acute (worsening) and non-acute hospitalizations , leading to wrong conclusions. There is a clinical difference between hospitalization due to HF and worsening HF. Unfortunately, no different ICD-10 codes are available for chronic and acute HF provided for chronic and acute coronary syndromes. Thus, differentiation between urgent and non-urgent HF hospitalization may be difficult or impossible in retrospective studies. Moreover, in studies on myocardial infarction, non-fatal myocardial infarction was widely used as an endpoint and an element of a composite endpoint of death or non-fatal myocardial infarction. Such an endpoint probably cannot be used in HF, as it may be difficult to distinguish between a non-fatal acute HF and non-fatal chronic HF hospitalization.In real-life settings, excessive reduction in HF rehospitalizations may increase mortality. Such an observation was made in the USA’s Medicare population, where hospitals with the worst hospital readmission rates were penalized. Consequently, the reduction in hospital admissions and economic profits was obscured by the higher mortality, as patients died outside the hospital . MoreoveOn the other hand, the composite endpoint “death or non-fatal hospital readmission” favors mortality over rehospitalization as the composite endpoint’s element compared to the “death or hospital readmission”. According to the composite endpoint definition, the first registered event of a composite endpoint is considered the endpoint. In consequence, when death occurs during the hospital stay, the first endpoint  will be counted as death and the second  as hospital readmission. The second issue is a time-to-event difference, which is supposed to be longer in the “death or non-fatal hospital readmission” endpoint, as patients who die in a hospital will be censored later. According to the Heart Failure Association of ESC statement, the preferred outcome in terms of mortality is cardiovascular mortality . Among 3In our opinion, the newly defined endpoint may have two practical applications. Our study showed that it could be easily calculated in most published trials. In such a case, it might explain the mechanism of death, as patients without rehospitalization before death most likely died of sudden cardiac death or were palliative patients who were not hospitalized and died at home. For that reason, it might help to interpret the outcomes of both positive and negative trials and potentially explain why some trials did not reach their goals in terms of the composite endpoints or hospital readmission. The conventional endpoints might not reveal all aspects of patient outcomes. Some therapies might reduce the death rate without previous hospital admission, which could be beneficial, even without reducing overall long-term mortality. Therefore, the second potential application of the newly defined endpoint may be in future clinical trials in different populations, including patients with heart failure, myocardial infarction, or after ICD/CRT-D implantation, to provide additional data on the structure of the composite endpoint. We do not intend to change or replace conventional hard endpoints with the newly defined endpoint as a primary endpoint but to deliver more detailed information on the outcome.Our study has some limitations. Trials included in the analysis had different enrolment criteria and were conducted between 1986 and 2020, which affected individuals’ baseline characteristics, including drugs and device therapies . MoreoveTo sum up, the definition of endpoints in clinical trials plays a pivotal role in interpreting trial outcomes, affecting the clinical guidelines and recommendations. The new endpoint describing patients who died without prior hospital admission might be useful in previous and future interventional studies to provide additional data on the structure of the composite endpoint. Some therapies might reduce death without previous hospital admission rates, which could be beneficial, even without a reduction in overall long-term mortality.


In recent years, accumulating evidence from translational research has highlighted the significant role of the gut microbiota and its metabolites in shaping the cardiovascular and cardiometabolic phenotypes. In particular, combined metagenomic and metabolomic approaches have contributed to our understanding of how intestinal microbial metabolism of dietary nutrients impact metabolic pathways and cardiovascular disease risk , 2.A number of metaorganismal circuits have been reported to modulate different aspects of cardiovascular and cardiometabolic conditions, such as arterial hypertension and hypertensive end organ damage , 4, lipiFrontiers in Medicine Research Topic “Impact of the Gut Microbiota on Cardiovascular Medicine” we highlight research and review articles presenting novel insights into the interaction between the gut microbiota and cardiometabolic and cardiovascular (patho-)physiology.In the current article collection on the Lang et al. provide novel evidence of how manipulation of plasma lipid levels modulates the gut microbiome. In particular, manipulated plasma lipid levels of non-human primates using dietary and pharmacological techniques, and characterized the microbiome using 16S rRNA gene sequencing. Their study showed that high-fat diets significantly reduced alpha diversity (Shannon) and the Firmicutes/Bacteroidetes ratio compared to chow diets, even when the diets had different compositions and were applied in different orders. Pharmacological manipulation of plasma lipid levels through parenteral means however, using antisense oligonucleotides, did not affect the microbiome composition. Moreover, they demonstrated that liver X receptor (LXR) agonist treatment shifted the microbiome. Fifteen genera increased with the LXR agonist, while seven genera decreased. For example, Pseudomonas increased upon LXR agonist treatment and was negatively correlated to deoxycholic acid, cholic acid, and total bile acids while Ruminococcus was positively correlated with taurolithocholic acid and taurodeoxycholic acid. Most bile acids identified in the feces significantly decreased in response to LXR agonist treatment, and total bile acids were reduced by 62%. These results emphasize that bile acids, derived in part from plasma lipids, are likely responsible for the indirect relationship between plasma lipid levels and the microbiome. Since bile acids are toxic to bacteria, they appear to considerably shift the microbiome composition. Thus, the indirect relationship between microbiome and plasma lipid levels is most likely mediated by bile acids.Gesper et al. demonstrate a functional link between the gut-derived metabolite indole-3-propionic acid and mitochondrial function in cardiomyocytes and how this metabolite alters cardiac function. First, the authors screened 25 microbial metabolites in a live-cell metabolic assay using HL-1 cardiomyocytes. They identified indole-3-propionic acid, a microbial tryptophan derivative, as a modulator of mitochondrial function. While acute treatment induced enhancement of maximal mitochondrial respiration, chronic exposure led to mitochondrial dysfunction. In isolated perfused mouse hearts, indole-3-proprionic acid was shown to dose-dependently improve cardiac contractility. Since indole-3-propionic acid also reduced maximal respiration in cultured endothelial and hepatocyte derived cellular carcinoma cells, the revealed effect of this microbiota-derived tryptophan derivative is not restricted to cardiac tissue. Cumulatively, their results indicate a direct impact of microbial metabolites on cardiac physiology.In another study Ding et al. present the study design of a prospective observational case–control study to analyze the variation in the intestinal microflora and metabolites in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and to observe the outcomes of patients with routine clinical interventions. This study is of particular interest as in the critical care unit, most common clinical interventions such as enteral feeding, proton-pump inhibitors, systemic catecholamines and systemic antibiotics change the intestinal microbiome composition. Since in critically ill patients, changes in the intestinal flora and metabolites co-occur with systemic inflammatory reactions and often bacterial sepsis, the authors hypothesize that intestinal dysbacteriosis after CPB, enhanced by intestinal ischemia-reperfusion injury, is a possible mechanism of inflammation and that bacterial translocation contributes to the development of sepsis.Chen et al. summarize current knowledge about antihypertensive drug-microbiota interactions and discuss how these interactions may help to develop gut microbiota-based personalized concepts for disease management, including antihypertensive response biomarkers, microbial-targeted therapies, and probiotics therapy.Finally, These contributions shed further light into the interaction between the gut microbiome and (patho-)physiolgogical pathways in the cardiovascular system and highlight the potential of microbiota-related strategies to improve currently available diagnostic and treatment options in cardiovascular medicine.AH wrote the first draft of the manuscript. TvM, GP, and CR wrote sections of the manuscript. All authors read and approved the submitted version.CR is a fellow of the Gutenberg Research College at the Johannes Gutenberg-University Mainz, a scientist at the German Center for Cardiovascular Research (DZHK), Partner Site Rhine Main, and acknowledges funding from the Forschungsinitiative Rheinland-Pfalz and ReALity. AH is participant in the BIH-Charité Advanced Clinician Scientist Pilotprogram funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Many inflammatory diseases that are responsible for a majority of deaths are still uncurable, in part as the underpinning pathomechanisms and how to combat them is still poorly understood. Tissue-resident macrophages play pivotal roles in the maintenance of tissue homeostasis, but if they gradually convert to proinflammatory phenotypes, or if blood-born proinflammatory macrophages persist long-term after activation, they contribute to chronic inflammation and fibrosis. While biochemical factors and how they regulate the inflammatory transcriptional response of macrophages have been at the forefront of research to identify targets for therapeutic interventions, evidence is increasing that physical factors also tune the macrophage phenotype. Recently, several mechanisms have emerged as to how physical factors impact the mechanobiology of macrophages, from the nuclear translocation of transcription factors to epigenetic modifications, perhaps even DNA methylation. Insight into the mechanobiology of macrophages and associated epigenetic modifications will deliver novel therapeutic options going forward, particularly in the context of increased inflammation with advancing age and age-related diseases. We review here how biophysical factors can co-regulate pro-inflammatory gene expression and epigenetic modifications and identify knowledge gaps that require urgent attention if this therapeutic potential is to be realized. I.Despite the significant progress made in medicine, we still struggle today with treating many long term, progressive diseases that are typically accompanied by chronic inflammatory processes. As the clinical focus historically has been on organ-specific diseases, medicine was compartmentalized along organ-specific divisions, which presents challenges to recognizing generic pathogenic mechanisms. Even though organ-specific cell niches have distinct biochemical and biophysical characteristics, inflammatory processes are at the core of many chronic diseases and common pathomechanisms are now emerging. The importance of understanding the role of biomechanical signals in the inflammatory status of cells, including macrophages and fibroblasts, is exemplified by the impact of inflammation in human ageing and age-related disease. Advancing age is accompanied by an increase in systemic inflammation, termed inflammageing,in vitro cell culture experiments, as they mimic the interaction of immune cells, including macrophages, with the cell wall of gram-negative bacteria. Macrophages, the major cellular drivers of the inflammatory process, originate from two distinct lineages:The term “inflammation” refers to a cascade of events that often starts with an infectious challenge or a sterile injury that activates immune cells, notably macrophages, to produce a range of soluble mediators (cytokines) that mediate the immune system response. Eventually, this inflammatory response is actively terminated to ensure homeostasis and the healing of tissue, i.e., “resolution of inflammation.”While niche-specific transcriptomic and epigenetic profiles as well as secretome are well documented for multiple tissues and various physiological contexts, cell phenotypes are also determined by a range of biophysical factors and stimuli.In contrast, current research in immune cell biology is still dominated by a cell centric view, rather than taking a more holistic view that considers how cells react to both biochemical and biophysical microenvironmental factors. With respect to immune cells, T cell activation is affected by substrate stiffnessDespite this rapid emergence of mechanobiology and the study of underpinning mechanisms, knowledge gaps exist as to how these mechanisms relate to chronic inflammatory diseases and, in particular, the relevance to inflammatory macrophages. How to intervene to reestablish homeostasis after infection, injury or pathological tissue transformation is still one of the biggest challenges of regenerative medicine. As the field of immunology has only recently started to appreciate the impact of physical factors on macrophage phenotype regulation, a review of the literature is provided here to summarize what is known of how biophysical factors can regulate inflammatory gene expression and macrophage polarization in the context of inflammation and disease, and what research questions require urgent attention. Current evidence highlights already how the changing landscape of biomechanical signals during inflammation directs several inflammatory biochemical intermediates in response to pro-inflammatory activation, which then converge onto transcriptional and epigenetic modifications (histone modifications and DNA methylation) and changes in the chromosome landscape as reviewed here. As such these changes could be either modulated by altering the biophysical properties of the microenvironment or be targeted by specific drugs modulating mechanosensitive signals. Since these nuclear events and epigenetic modifications have previously been shown in other cell types to be dependent on the nucleoskeleton or nuclear meshwork, the data taken together also suggest the existence of an unknown and yet to be explored role of the nuclear meshwork in driving inflammatory activation of macrophages, which requires urgent attention. Even though recent reviews have individually provided a glimpse onto these regulatory events,II.Organ-specific cell niches are complex and comprise many cell types, all in close proximity to each other. Each organ is characterized by organ-specific cell types that are supported and surrounded by stromal cells common to most organs, including fibroblasts as well as immune cells that have sentinel functions in sensing injury and infection. This latter group includes primarily tissue resident macrophages and macrophages derived from activated, homed-in circulating macrophages. Other common cellular tissue residents can include adipocytes as active endocrine producers, as well as perivascular mesenchymal stem and endothelial cells. These cell societies communicate with each other biochemically and physically. They respond to global metabolite compositionCell niches are not only characterized by the resident cells but also have characteristic ECM compositions, assembled into complex meshworks of nanofibrils that are undergoing constant remodeling by the synergistic actions of various cell types. Some of these cells synthesize the ECM or its components, while they themselves or others contribute by secreting cross-linking and proteolytic enzymes.III.More than a century ago, it was first reported that brain-resident macrophages, i.e., microglia, have a characteristic morphology defined by a small cell body with fine ramified processes , left.90As for tissue resident macrophages, also the pro-inflammatory activation of bone marrow derived macrophages (BMDMs) with LPS leads to major morphological changes: with a delay of a few hours, the cell spreading area increases significantly and the macrophages transition from an elongated to a more rounded cell shape ,12 whichIV.IL-6, iNOS, CXCL9), necessary for prolonging the process of inflammation, is dependent on macrophage spreading.With the advent of bioengineering tools and techniques, we can now address in detail the key question: Does macrophage activation first lead to a change of gene expression profile which is then followed by a change in cell morphology, or vice versa? Recent data show that certain cell morphologies, as imposed by spatial constraints, can help in stabilizing macrophage phenotype. To ask whether staging a fully fledged inflammatory response requires actin polymerization, bioengineered tools were used to spatially confine macrophages on micropatterned adhesive islands of defined sizes and shapes. For example, the production of inflammatory cytokines including IL-6 and tumor necrosis factor-alpha (TNF-α) was greatly reduced when the spreading of BMDMs was restricted by culturing them on adhesive islands of appropriate size, in hemispherical pores, or in a close-packed cell layer.in vitro,These findings are significant, as tissue resident macrophages are found in a range of tissue-specific sizes and shapes, surrounded by niches with tissue-specific characteristics including stiffness, ECM composition, fiber density, and fiber tension.V.Changes in cellular morphology are coupled with a reorganization of the cytoskeleton. As the first fragile contacts, i.e., focal adhesions, become mechanically reinforced, assembly of a mature cytoskeleton progresses rapidly, guided by the spatial locations of the point of contact to the extracellular matrix. Major insights into these processes were gained via research on mesenchymal cells through the micro- and nanofabrication of adhesive contacts. These studies illustrated how the cytoskeletal filaments are bundled along with the primary directions of force transmission through the cell, as imposed by the spatial presentation of environmental anchor points.Initiating actin polymerization involves the phosphorylation of paxillin on tyrosine 118 and Neural Wiskott-Aldrich syndrome protein (N-WASP) on serine 484/485, two actin-regulatory proteins important for actin polymerization and reorganization.VI.IL-6 and Nos2 IL-6 and While the activity of most of these transcription factors during pro-inflammatory activation was characterized downstream of toll-like receptor 4 (TLR4) signaling, the major receptor that mediates LPS signaling,Contrary to pro-inflammatory macrophage activation, the organization and levels of actin polymerization during pro-healing M2 macrophage activation are still not well characterized. Even though certain reports suggest that inhibiting actin polymerization and myosin contractility significantly reduce M2 activation, without altering their cell elongation,VII.As cytoskeletal components mechanically couple the cell adhesions of the plasma membrane to the cell nucleus, the nucleus can get mechanically strained leading or at least contributing to its remodeling. In fibroblast and epithelial cells, highly tensed actin stress fibers, called the actin-cap,In contrast to mesenchymal cells, blood born cells often must crawl through tiny constrictions to reach their destiny, including transmigration through the endothelium, which requires that they undergo extreme cellular and nuclear deformations.VIII.As cell size and nuclear size are positively correlated, it has been shown that pro-inflammatory M1 activation of macrophages results in a significant increase in the nuclear projection area and nuclear volume.in vitro and in vivo), there is a significant decrease in the levels of lamin-A/C mRNA (IL-6 and TNF-a) and also pro-inflammatory cytokine secretion. Regarding tissue-resident macrophages, lamin A/C ablation in immune cells results in a selective depletion of lung alveolar macrophages and a heightened susceptibility to influenza infection.Differentiation of rat and human macrophages has been shown to be accompanied by increased expression of lamin-A/C,A/C mRNA . ConcomiDownregulation and degradation of the nuclear lamina are thus a mechano-regulated process and depend on a variety of physical parameters, including substrate stiffness.IX.Expression of the pro-inflammatory secretome requires access to the corresponding genes. It is worth mentioning that the changes in accessibility of chromatin and genes, as probed using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq), has shown that accessibility depends on the mechanical environment and mechanical status of cells. Uniaxial cyclic stretching of MSCs, for example, induces differentiation into osteoblasts by upregulating the chromatin accessibility of genes associated with the regulation of cell morphogenesis, cell–substrate adhesion, and ossification.During the past decade, genome-wide mapping methods have identified genomic regions that are in close contact with the nuclear lamina, termed lamina-associated domains (LADs).Nuclear lamina-influenced changes in gene expression are also associated with changes in gene positioning. For example, knockdown of lamin A/C deregulates expression levels of genes; both KLK10  and MADH2  were significantly repressed, while BCL2L12  was de-repressed.Drosophila S2 cells also leads to chromatin compaction and repositioning from the nuclear envelope.Finally, it has been shown in several cell types that changes in the nuclear lamina and in lamin-A/C aid the repositioning of chromosomes due to altered interactions between chromosomes and the inner nuclear membrane. Three-dimensional DNA-immunoFISH revealed that the repositioning of chromosomal regions to the nuclear lamina is dependent on breakdown and reformation of the nuclear envelope during mitosis. During mitosis, chromosome movement correlates with reduced lamin association with the nuclear rim.X.Epigenetics is the study of how cells control gene activity without changing the DNA sequence.The first evidence of the link between LPS stimulation and epigenetic regulation in inflammatory genes dates back to 1999, as LPS stimulation induces the cytokine IL-12p40 production in murine macrophages by rapid and specific nucleosome translocation at the promoter region.μm in width and spacing) showed not only markedly increased global AcH3 marks, but also a significant increase in methylation (both di- and tri-methylation) of histone H3 at lysine 4  relative to flat surfaces.μm wide).Various histone modifications in response to alterations of physical properties of the cell microenvironment have recently been reported for mesenchymal cells. Acetylation of H3 on Lysine 9 (AcH3K9), for example, depends on the cell spreading area of fibroblasts.In contrast, whether histone modifications induced during macrophage activation are cell niche dependent was not known until recently . H3K36meIL-6.Histone modifications are largely driven by histone methyltransferases (HMTs), histone acetyltransferases (HATs), and histone deacetylases (HDACs), which have been extensively reviewed elsewhereDNA methylation, together with histone modifications, can also regulate gene expression.in vitro and in vivo blood flow models revealed that disturbed flow, as observed during atherosclerosis and characterized by low and oscillating shear stress, induces expression of DNMT1 and thereby regulates the genome-wide DNA methylation pattern. However, whether the tissue-dependent mechanical properties of microniches as discussed above drive macrophage homeostatic function and inflammation via DNA methylation remains unknown. As cells cannot only feel the stiffness of their microenvironment but also respond to the stretch-induced switching of the functional display of ECM fibers,Whether DNA methylation is a mechano-regulated process remains elusive . Only reFinally, non-coding RNAs (miRNAs and lncRNAs) are important regulators of epigenetic modifications and, thus, gene expression, and their regulatory role during macrophage polarization has only recently been explored.XI.Physiological ageing is accompanied by a chronic, sub-clinical increase in pro-inflammatory cytokines  and reduced anti-inflammatory cytokines (IL-10) in the blood, termed inflammageing.While inflammageing is driven by many factors, a key contributor is the appearance of monocytes/macrophages that are in a state of low-level constitutive activation, resulting in the secretion of pro-inflammatory cytokines in the absence of infection.In a variety of cell types, there is increasing evidence that ageing is associated with changes in the mechanical properties of cells and strong correlations exist between age and cytoplasmic stiffness.Other important contributors to inflammageing include senescent cells. These proliferatively quiescent cells are highly metabolically active, producing a complex pro-inflammatory secretome termed the senescence associated secretory phenotype (SASP).6-integrin is a matrix stiffness-regulated mechanosensitive molecule, which confers an invasive fibroblast phenotype.As stated earlier, it is now widely recognized that most age-related diseases have a strong inflammatory component. The source of this inflammation is varied ranging from increased adiposity to reduced physical inactivity but includes the increased pro-inflammatory status of macrophages and cell senescence.XII.It is of uttermost importance to find cures for the many inflammatory diseases that are responsible for the majority of deaths and whose incidence increases with age. What has become evident is that an improved understanding of the role of mechanical forces in modulating the inflammatory status of cells such as macrophages and senescent stromal cells will deliver novel therapeutic options going forward. Creating new paradigms, which integrate biochemical, immunological, and mechanobiological factors, will produce significant new insights into age-related disease pathogenesis. Defining how to regenerate tissues affected by inflammatory pathologies, a major challenge in regenerative medicine, also requires mechanobiological knowledge. Since the demonstration that substrate stiffness correlates with mesenchymal stem cell differentiation fate,


A six-month-old female presented to a community hospital with small lacerations to the scalp, face, and left eyelid from a dog bite injury. Computed tomography imaging revealed an impacted right frontal bone fracture and left superior orbital fracture, prompting transfer, neurosurgical repair, and infectious disease management of the injury.This report highlights the importance of having a high level of suspicion for deeper injury in pediatric and especially infant craniofacial dog bites, obtaining radiographic imaging, and initiating appropriate multidisciplinary triage to prevent life-threatening infection and complications. A six-month-old female presented to a community hospital with superficial-appearing lacerations of the scalp, face, and left eyelid after a dog bite. The dog and infant were both up to date on immunizations. Upon presentation, the patient was alert and stable. Computed tomography imaging demonstrated right frontal impacted calvarial fracture with displaced fragment and adjacent right frontal intraparenchymal hemorrhagic contusions, as well as a left superior orbital fracture . The patPasteurella canis, Viridans streptococci, Staphylococcus aureus, and Fusobacterium. Neurosurgery took the patient to the operating room for irrigation and debridement and elevated and repaired the depressed skull fracture, contouring the calvarium to its normal frontal shape. Plastic surgery repaired the remaining facial and eyelid lacerations.Upon transfer, the patient was neurologically intact. The infectious disease (ID) team recommended treatment with ceftriaxone and metronidazole for central nervous system coverage and broad-spectrum coverage of common canine oral flora, including Following repair, the patient was admitted to the intensive care unit for monitoring for two nights and ultimately discharged on postoperative day five. According to ID recommendations, the patient was continued on ceftriaxone and metronidazole for four weeks post-surgery. The patient recovered without seizures, emesis, lethargy, fever, or weakness. Follow-up magnetic resonance imaging four months post injury showed evolving right frontal lobe encephalomalacia at the site of presentation of hemorrhagic contusions but no evidence of intracranial abscess, fluid collection, or parenchymal edema to suggest ongoing infection. The lacerations healed well.Although there are a handful of reports in the craniofacial and neurosurgical literatureOpen skull fractures and depressed skull fractures with underlying mass effect or aesthetic implications are both indications for neurosurgical washout and repair. Our standard management of such fractures entails prophylactic pre- and intraoperative antibiotics, as well as postoperative antibiotics for polymicrobial exposure. Whereas punctate bite wounds in soft tissue alone are often left open to drain and heal secondarily, such injuries over open skull fractures are typically closed after thorough irrigation, debridement, and fracture reduction. Follow-up imaging is obtained to rule out abscess development.What do we already know about this clinical entity?Infants are more susceptible to skull fractures from dog bite lacerations to the head.What is the major impact of the image(s)?Computed tomography imaging showed a depressed skull fracture under the superficial-appearing puncture wound. This finding necessitated surgical intervention.How might this improve emergency medicine practice?Beware of superficial-appearing scalp or facial dog-bite injuries in infants; imaging may reveal skull injuries that portend risk of life-threatening complications.We recommend that clinicians be wary of minor-appearing dog bite lacerations of the scalp in infants and strongly suggest obtaining imaging in the emergency department for all young patients with dog bites to the head.


The regulation of blood flow to peripheral muscles is crucial for proper skeletal muscle functioning and exercise performance. During exercise, increased mitochondrial oxidative phosphorylation leads to increased electron leakage and consequently induces an increase in ROS formation, contributing to DNA, lipid, and protein damage. Moreover, exercise may increase blood- and intramuscular inflammatory factors leading to a deterioration in endurance performance. The aim of this review is to investigate the potential mechanisms through which the polyphenol hesperidin could lead to enhanced exercise performance, namely improved endothelial function, reduced exercise-induced oxidative stress, and inflammation. We selected in vivo RCTs, animal studies, and in vitro studies in which hesperidin, its aglycone form hesperetin, hesperetin-metabolites, or orange juice are supplemented at any dosage and where the parameters related to endothelial function, oxidative stress, and/or inflammation have been measured. The results collected in this review show that hesperidin improves endothelial function (via increased NO availability), inhibits ROS production, decreases production and plasma levels of pro-inflammatory markers, and improves anaerobic exercise outcomes . For elite and recreational athletes, hesperidin could be used as an ergogenic aid to enhance muscle recovery between training sessions, optimize oxygen and nutrient supplies to the muscles, and improve anaerobic performance. During exercise, skeletal muscle cells convert biological fuel  into mechanical force to allow muscle contraction and therefore movement. The energy required for this motion is largely provided by the breakdown of adenosine triphosphate (ATP). Intramuscular stores of ATP can sustain only a short period of muscle activity. Therefore, ATP needs to be generated by anaerobic glycolysis and oxidative phosphorylation . During Blood flow is the main regulator of the skeletal muscles’ oxygen supply. Skeletal muscle contains a dense capillary network that serves to deliver oxygen and nutrients and remove waste products from the skeletal muscle cells . To ensu2O2) might be an important factor in the regulation of vascular tone by functioning as an endothelium-derived hyperpolarizing factor (EDHF) leading to vasoconstriction . O. O107]. Acute supplementation with 217 mg of hesperidin in healthy soccer players decreased the lipid peroxidation marker malondialdehyde (MDA) post-exercise in plasma . Plasma 2, NO, and NO2, and decreased protein levels of COX-2 and iNOS ..104].2max) [2max was recorded in the intervention group compared to the placebo. VO2max is defined as the maximum rate of oxygen consumption measured during severe exercise [2max is used to assess endurance performance and it is limited by the ability of the cardiorespiratory system to deliver oxygen to the exercising muscles [2max was recorded during a 10 min time trial on a cycle ergometer after 4 weeks of hesperidin (450 mg/day) supplementation [2/power ratio significantly improved in the intervention group compared to the placebo, resulting in a higher amount of power produced per unit of oxygen consumed (VO2/power ratio).Ingesting 500 mg of hesperidin 5h before a repeated sprints test (Wingate test) was able to improve anaerobic performance outcomes (average power (W); maximal speed (rpm); and total energy (J)) in the intervention group compared to the placebo  Table 1. Ingesti2max) . Howeverexercise . In exer muscles . No signentation . In the Martínez-Noguera et al. and Van Iersel et al. both tested the chronic effects of hesperidin supplementation on sport performance outcomes after a Wingate test ,117. SupThe studies collected in this review show the potential of hesperidin, hesperetin, and their metabolites to enhance exercise performance by (i) improving endothelial function  pumping in the sarcoplasmic reticulum (Ca2+-ATPase) [2+ release from the sarcoplasmic reticulum [2+-ATPase activity [In vitro studies investigating the effects of hesperidin, hesperetin, and their metabolites in endothelial cells highlight the potential of the flavanone to enhance the production of NO in the vascular endothelium. There is growing evidence showing that increased NO availability can improve exercise-related performance through enhanced tissue oxygenation (due to blood vessel vasodilation) combined with increased metabolic efficiency in active skeletal muscle . Increas-ATPase) . NO, beieticulum  and inhiactivity , can decactivity . When itactivity . TherefoThe studies collected in this review showed enhanced vasodilator responses after supplementation with hesperidin in both healthy and unhealthy individuals/animals. Although in subjects with hypertension, there is a different regulation in blood vessel vasomotor responses compared to healthy people and, therefore, athletes ,124. FutThe included studies were consistent in the ability of hesperidin and hesperetin to inhibit ROS production in a variety of cell types and tissues. Despite the broad amount of literature supporting the role of hesperidin in antioxidant cellular defences, there is still a lack of studies focusing specifically on its effects on skeletal muscles. More RCTs should be conducted to ascertain the effects of hesperidin on oxidative status after exercise . FurtherHesperidin and hesperetin showed good anti-inflammatory properties by decreasing the production and plasma levels of pro-inflammatory markers. Despite the evidence from studies performed on untrained and unhealthy subjects, we do not have enough data to support the role of hesperidin in restraining systemic inflammation in overtrained subjects. More research is needed to validate our hypothesis that the anti-inflammatory properties of hesperidin can lead to a reduction in intramuscular inflammation and muscle damage, and in this way result in increased exercise performance. Future studies should not only investigate the effects of hesperidin supplementation on systemic post-exercise inflammation markers but also evaluate the changes in intramuscular inflammation markers via skeletal muscle biopsies.Finally, the effects of hesperidin supplementation on improved exercise performance have been investigated. In rats, supplementation with hesperidin led to increased performance in maximal running distance. In trained athletes, both acute and chronic hesperidin intake was able to improve multiple anaerobic exercise outcomes . Further studies are needed to assess the effects of hesperidin supplementation on endurance exercise in humans.2) efflux to the muscle, which is associated with increased endurance performance. Moreover, hesperidin can decrease ROS-mediated damage in muscle cells, which enhances muscle function. Finally, hesperidin can decrease post-exercise-induced inflammation, which potentially speeds up the recovery process and can thereby improve exercise performance. In this way, personalized supplementation with hesperidin seems to increase anaerobic exercise performance, although further research is necessary to draw conclusions regarding the efficiency of hesperidin supplementation for endurance athletes.In conclusion, the ergogenic effects that hesperidin can bring to the spectrum of improved exercise performance are promising and should be investigated further. For elite and recreational athletes, hesperidin could be a promising food supplement to optimize the oxygen and nutrient supplies of the muscles, stimulate muscle contraction, and enhance muscle recovery between training sessions. During exercise, hesperidin supplementation can increase endothelial function, thereby contributing to increased skeletal muscle perfusion and increasing oxygen (O


Pre-clinical and clinical spinal cord injury (SCI) studies differ in study design, particularly in the demographic characteristics of the chosen population. In clinical study design, criteria such as such as motor scores, neurological level, and severity of injury are often key determinants for participant inclusion. Further, demographic variables in clinical trials often include individuals from a wide age range and typically include both sexes, albeit historically most cases of SCI occur in males. In contrast, pre-clinical SCI models predominately utilize young adult rodents and typically use only females. While it is often not feasible to power SCI clinical trials to test multi-variable designs such as contrasting different ages, recent pre-clinical findings in SCI animal models have emphasized the importance of considering age as a biological variable prior to human experiments. Emerging pre-clinical data have identified case examples of treatments that diverge in efficacy across different demographic variables and have elucidated several age-dependent effects in SCI. The extent to which these differing or diverging treatment responses manifest clinically can not only complicate statistical findings and trial interpretations but also may be predictive of worse outcomes in select clinical populations. This review highlights recent literature including age as a biological variable in pre-clinical studies and articulates the results with respect to implications for clinical trials. Based on emerging unpredictable treatment outcomes in older rodents, we argue for the importance of including age as a biological variable in pre-clinical animal models prior to clinical testing. We believe that careful analyses of how age interacts with SCI treatments and pathophysiology will help guide clinical trial design and may improve both the safety and outcomes of such important efforts. The average age at the time of spinal cord injury (SCI) has increased over time. In the 1970s, the average age at the time of SCI was 28 years old (yo) but as of 2015 has increased to 43 years old . DespiteIn this review, we will discuss why older age at the time of SCI is associated with worse functional outcomes in animal models as well as the confounding variables that affect the interpretation of age-dependent effects clinically. We will review several biological underpinnings of secondary injury and recovery that are affected by the aging process. Specifically, we will cover known physiological aging adaptations that influence SCI responses, exacerbate secondary injury, and worsen functional outcomes. We will also discuss the somewhat unpredictable and unexpected results of animal studies focusing on interventions tailored to treat age-associated physiological differences. The conclusion of our comprehensive evaluation, namely that age can have profound effects on treatment approaches, supports the re-evaluation of pre-clinical therapeutic strategies as well as indicates that the minimal information necessary to translate preclinical results into clinical trials should be reconsidered. Articles in this review were chosen based on a comprehensive review of pre-clinical animal literature covering comparisons between animal models of young adult ages and older, as well as selected clinical reports offering contrasting findings about the role of age in the SCI population.Determining how age at the time of injury affects clinical outcomes after SCI is challenging. Mortality after SCI increases with age creating a potential selection bias where more resilient, or less severely injured, older individuals are a larger representation within longitudinal clinical data . This biAs previously noted, the average age of SCI in the US has increased to 43 years old. The causes, spinal levels, and severity of SCI have also changed over time with the most frequent category of neurological injury being incomplete tetraplegia (48.6% in the US since 2015) . In the The difficulty in directly comparing outcomes between age groups in humans likely contributes to conflicting reports across retrospective studies examining age at the time of SCI . For exaFindings from the In contrast to Many caveats with interpreting clinical reports  can be addressed through controlled modeling in animals. Comparing between younger and older rodents in pre-clinical SCI models has elucidated several underlying differences occurring with advancing age in the pathology and recovery following SCI. Specifically, when injury severity, anatomical location, and injury type are controlled across age groups in rats and mice, older age is associated with worse functional outcomes, even when comparing young adults (3–4 months old) to middle-aged (12–14 month-old) rodents . The abiAn age-associated decrease in locomotor outcomes has been replicated across labs and in both mice and rats. In this review, we will not discuss differences between neonatal/juvenile/pediatric and adult ages but will limit discussion to differences between young-adult, middle-aged, and elderly groups. The majority of reports from rodent studies examining the effects of age at the time of SCI reproducibly demonstrate that older age results in worse functional outcomes. In rats, age-associated impairments in functional recovery have been demonstrated using the Basso, Beattie, Bresnahan scale of locomotor recovery (BBB) . SimilarIn addition to locomotor recovery, older age at the time of SCI is associated with differences in sensory function in rodents . Use of Several studies identify that functional recovery is indeed reduced in older rodents after SCI, however, the biological mechanisms that underlie decreased recovery continue to be explored. The aging central nervous system is well-known to possess a decreased capacity for plasticity and regeneration, particularly in the context of hippocampal memory formation . HippocaTo date, there have only been three published reports evaluating the effects of advanced age on regeneration after SCI, with all three reports providing corroborating evidence for observations in other neurological conditions . Specifiin vivo after both optic nerve crush and SCI  leads to an exacerbated intraspinal inflammatory response. Specifically, we observed a loss of IL-4 receptor (IL-4R) on microglia and macrophages in 18-month-old mice after SCI . IL-4R sfter SCI . We obsefter SCI . Subsequfter SCI . The profter SCI .Older age at the time of injury is also associated with increased recruitment of macrophages into the lesion in both rats and mice . We alsoTwo other recently published manuscripts provide more evidence highlighting the importance of inflammation in age-dependent effects after SCI. First, in vivo after SCI, as well as attenuate pro-inflammatory signaling in macrophages and mitigate macrophage accumulation within SCI lesions (Targeting inflammation by knockout out micro-RNA-155 (miR-155) has also been used as a strategy to determine the effects of inflammatory signaling on pain development in older age (2- vs. 20-month) animals after SCI . miR-155 lesions . miR-155 lesions , mitigat lesions . Regardl lesions . The colRedox metabolism is known to change with advanced age and resembles a shift toward a stronger reliance on glycolysis for energy production, a term coined the Warburg effect which was originally identified in cancer  and is e2  can induce a mild-uncoupling response that aids in reducing ROS produced by mitochondria , a dye sensitive to superoxide formation, reveals an age-dependent increase in active ROS production at both 3- and 7-dpi . DHE is While the capacity to defend against free radicals is quite complex, one major cellular pathway, the glutathione (GSH) system, has been investigated for its known changes occurring with age . CellulaGlutathione depletion after SCI occurs as early as 24-h post-SCI and remains depleted for up to 72-h, and likely longer, with greater decreases at 72- compared to 24-h post-injury . Interesn-acetylcysteine amide (NACA) which has better bioavailability within the spinal cord  or a more recently developed nal cord . Treatinnal cord . Owing tnal cord , we prednal cord . While tWhile the effects of aging on inflammation, mitochondrial function, and redox metabolism have been well-characterized in many physiological systems, there are other consequential interactions between age and SCI not directly related to central pathology. Similar to clinical findings, older mice experience greater mortality after SCI . FindingThe consequences of being older at the time of injury are not just associated with an increase in unexpected death but are also associated with other measures of morbidity such as weight loss. Older rats and mice lose more weight after SCI as a percentage of body weight compared to younger rats and mice . NotablyThe cause of increased mortality in older male mice remains unknown, but our observations point to a potential hematologic contributor. We recently reported that 14-month male mice appeared colder to the touch in the days following SCI and that 14-month male mice had noticeable less red blood cells (RBCs) in the blood after spinning down plasma compared to 4-month mice . SCI indFinally, a recent study by While work including age or sex as a biological variable is amassing, it remains important to recognize that males and females do not biologically age the same. The influence of sex hormones and how they change with age can give rise to reciprocal effects on several biological processes. For example, estradiol, which is believed to exert neuroprotective effects in SCI, sharply declines during menopause in humans . Not onlSimilar to humans, testosterone levels in rodents decreases with advancing age . While tThus far, we have identified several sex-by-age interactions which validate that some age-dependent injury responses are sex-specific. Specifically, the example provided above that identified a sex-by-age interaction of RBC/Plasma ratio is indicative of how aging can differentially affect outcomes in a sex-specific manner . FurtherWhile a consensus across several labs has concluded that older age reduces functional recovery after SCI in rodents, it remains important to highlight a few potential caveats that exist in animal modeling. First, it is impossible to test the same cohort of mice at two different ages at the same time, resulting in the use of different cohorts to represent differences in age. In some cases, the challenge associated with obtaining older mice has resulted in experimental strategies which utilize either retired breeders from animal colonies or the use of mixed populations of young and old mice from different colonies such as those purchased from the Jackson laboratories, Charles River, and the National Institute of Aging (NIA) aged animal repository. Utilizing animals from different colonies has an inherent potential to influence the aging process and SCI pathophysiology in unanticipated ways. For example, the C57BL/6 mouse lines have developed different but known mutations within the Charles River and Jackson Laboratory colonies. C57BL/6N mice from Charles River carry a recessive mutation in the Crb1 gene known to induce ocular lesions that impair visual perception in homozygous mice from the breeding colony . In contAnother complication with including different ages is the role of anatomical growth both of the animal as a whole, as well as the spinal cord in particular. Mice and rats gain weight with age, which can confound the interpretation or analysis of results. Of significant interest is an increase in spinal cord size and diameter with aging ,b. FourtBaseline differences are not simply limited to anatomical discrepancies that develop with aging. Baseline differences in functional outcomes, such as sensory thresholds and measures of forelimb strength, are reported to decline with older ages in uninjured rodents . In threFinally, while mortality can be mitigated to a degree in animal models, as reported above, several reports have detected an increased early death of mice and rats after SCI at older ages . ObserviTo summarize key findings from this literature review, age as a biological variable effects SCI injury and recovery processes as well as responses to treatments in often unpredictable ways. The average age at time of injury has increased to a mean age of 43 which creates a need to better understand the role of age in the SCI pathophysiology. There are several differences in the etiology of SCI between younger and older clinical demographics, the most pronounced of which is the primary mode of injury is caused by a higher prevalence of slip-and-fall accidents at older age. Slip-and-fall accidents are hallmarked by less severe conditions, often manifesting in central cord syndrome-like pathology. Differences in the primary mechanism of injury and/or severity of the initial insult makes comparing outcomes throughout the spectrum of age challenging. Indeed, several clinical reports implicate older age at the time of SCI as a variable which negatively impacts recovery, however after accounting for confounding variables, the impacts of age are less clear. The use of animal models to better understand the recovery potential throughout the spectrum of age has reproducibly determined that older age limits the recovery of motor functions after SCI. Mechanisms underlying a diminished functional recovery after SCI at older ages have implicated a reduced capacity for axon growth and regeneration in both non-intervened and intervened conditions, as well as more severe secondary injury cascades. Aging affects the acute pathophysiology of SCI through changes occurring at the level of inflammation as well as subcellular microenvironments. Macrophages which infiltrate the lesion after SCI display more aggressive and pro-inflammatory phenotypes that generate more reactive oxygen species at older ages. Within the cell, mitochondria accumulate age-related dysfunctions even prior to SCI that result in the generation of more oxidative stress as well as a reduced capacity to buffer cytosolic calcium. Oxidative damage is increased in the sub-acute stages of SCI at older ages, which can be explained likely by a combination of both an increase in the production of reactive oxygen species and a decrease in antioxidant defense. Most intriguingly, treating older mice after SCI with several antioxidant-based strategies has resulted in outcomes which would either be predictive based on the underlying biology, or completely counterintuitive to the logical hypothesis. The examples provided in this review emphasize an emerging pre-clinical notion that age at time of SCI can unpredictably affect treatment responses.Using animal models of SCI, we have identified a precedence for considering age as a biological variable in both pre-clinical and clinical research studies aiming to develop treatment approaches. The underlying pathophysiology of SCI changes with age in meaningful ways and treating those biological maladaptation’s yields unpredictable results. We have, thus far, identified treatment approaches that display larger and smaller treatment effects in older animals, as well as opposite effects to those observed in younger rodent counterparts. While we elucidate how aging affects different elements of the pathophysiology of SCI, we are finding that treatment approaches might not act the same throughout the spectrum of age. Most interventions acting on biological targets often have multiple effects, some beneficial to recovery and others detrimental, and changing the balance of these nuanced effects can influence the outcomes of treatment efforts. It is not difficult to imagine models in which a single molecular target can both promote recovery and damage, and to identify how changes in a system with older age can alter the net response to treatment . FollowiAS and JG: conceptualization and funding acquisition. JG: supervision. LJ: writing of the clinical aspects. All authors: writing and approving the submitted version.


The potential of seed priming is still not fully exploited. Our limited knowledge of the molecular dynamics of seed pre-germinative metabolism is the main hindrance to more effective new-generation techniques.Climate change and other recent global crises are disrupting food security. To cope with the current demand for increased food, feed, and biofuel production, while preserving sustainability, continuous technological innovation should be provided to the agri-food sector. Seed priming, a pre-sowing technique used to increase seed vigor, has become a valuable tool due to its potential to enhance germination and stress resilience under changing environments. Successful priming protocols result from the ability to properly act on the seed pre-germinative metabolism and stimulate events that are crucial for seed quality. However, the technique still requires constant optimization, and researchers are committed to addressing some key open questions to overcome such drawbacks. In this review, an update of the current scientific and technical knowledge related to seed priming is provided. The rehydration–dehydration cycle associated with priming treatments can be described in terms of metabolic pathways that are triggered, modulated, or turned off, depending on the seed physiological stage. Understanding the ways seed priming affects, either positively or negatively, such metabolic pathways and impacts gene expression and protein/metabolite accumulation/depletion represents an essential step toward the identification of novel seed quality hallmarks. The need to expand the basic knowledge on the molecular mechanisms ruling the seed response to priming is underlined along with the strong potential of applied research on primed seeds as a source of seed quality hallmarks. This route will hasten the implementation of seed priming techniques needed to support sustainable agriculture systems. To support such an impressive expansion—in line with the current global demand for increased food, feed, and biofuel production—continuous technological innovations should be provided to seed technologists, breeders, and farmers. There is strong pressure on these issues also because climate change is disrupting food security at the global level, a condition that has been further exacerbated by the coronavirus pandemic. The impact of COVID-19 on the production of certified seeds has been assessed by international institutions committed to developing strategies to preserve seed availability, accessibility, and quality (www.fao.org/family-farming/detail/ar/c/1331528/). The adverse effects of COVID-19 include a decreased production of seeds with certified quality due to the limited accessibility to the companies that supply certified seeds, along with challenges related to seed mobilization and field-based inspections  impacts crop physiology at different levels, including the occurrence of oxidative damage, decreased photosynthetic efficiency, and accelerated senescence  have been suggested to alter carbon/nitrogen ratios, with consequences on nitrogen availability , lentils (Lens culinaris Medik.), and fava beans (Vicia faba L.), with substantial losses in terms of yields and nutrient content , wheat , rice , and legumes, such as chickpea  and soybean  , cassava (Manihot esculenta L.), maize, oil palm (Elaeis guineensis L.), rapeseed (Brassica napus L.), rice, sorghum (Sorghum bicolor L.), soybean, sugarcane (Saccharum officinarum L.), and wheat. According to this study, the impact of global climate change on crop yields ranged from -13.4%  to 3.5% (soybean), with variable geographical distribution. A negative impact was observed in Europe, Southern Africa, and Australia, mixed situations were reported in Asia and Northern and Central America, whereas Latin America was characterized by positive effects. Such a scenario has already led to a reduction in consumable food calories in these crops  seeds soaked in milk or water before sowing resulted in faster germination . The Roman naturalist Gaius Plinius Secundus  underlined in his Naturalis Historia the relevance of presoaking seeds to improve germination  in which seeds were soaked for 2 days in manure water and then dried in the shade before sowing. Even Charles Darwin (1809–1882) conducted several experiments on seed germination, resulting in observations that contributed to his theory on the evolution of living organisms , explored different aspects of seed biology, from seed production to germination and conservation Evenari , 2007, o Evenari . The Frems Black . He testms Black . The seems Black .Phaseolus vulgaris L.) and pea (Pisum sativum L.) seeds before sowing. They also underlined that, despite this evidence, gardeners were still soaking their pea and bean seeds "to help them", taking advantage of their empirical ability to adjust the proper amount of water (Kidd and West Solanum lycopersicum L.) seedlings was improved when seeds were treated with solutions containing different salts . Heydecker and his coworkers imbibed the seeds of several horticultural and ornamental crops using a solution of the osmotic agent polyethylene glycol (PEG), obtaining accelerated and uniform germination  seeds , potassium phosphate (K3PO4), potassium chloride (KCl) salts, NaCl, CaCl2, MgSO4, or polyethylene glycol (PEG) with different water potentials, applied for different periods , and plant extracts. Their efficacy to enhance salt tolerance has been reported in different crop species, and mechanisms underlying the impact of these priming agents on the seed pre-germinative metabolism are also documented  have been documented  and in kidney beans subjected to salt stress , as well as cauliflower seeds and seedlings challenged with salt stress  to increase germination under high temperatures  seeds resulting in improved yield and percentage of seed oil in the field  seeds that underwent heat priming for 6 h at 35 °C displayed beneficial effects, including synthesis of osmolytes and increased photosynthetic performance, particularly evident in heat-sensitive genotypes  when applied to soybean and canola seeds under salt stress  resulted in beneficial effects on biochemical, morphological, and physiological characteristics of coriander (Coriandrum sativum L.) plants under Cd stress , prepared via co-precipitation with the marine macroalga Turbinaria ornata and used as a priming agent, could enhance seed germination in rice and maize   increased crop yield and nutritional quality with the added value of biosafety, as Ag did not bioaccumulate in edible tissues  of metal oxides, widely used in industries, appear as emerging tools for seed priming purposes in agriculture. The application of selenium and zinc oxide nanoparticles  during Chlorella vulgaris were used to produce AgNPs showing antimicrobial activity against Bacillus sp., Erwinia sp., Candida sp., and effective seed priming in Triticum vulgare and P. vulgaris  peel extract as reducing agent) were used as nanopriming agents for watermelon (Citrullus lanatus) seeds. This eco-friendly and sustainable nanotechnological approach enhanced seed germination, growth, and yield while maintaining fruit quality , selenium oxyanion-calcium hydroxide SeO-(Ca(OH)2), and selenium-calcium hydroxide Se-(Ca(OH)2) nanocomposites were tested in V. radiata seeds. Seed extracts from Cassia angustifolia, rich in galactomannan and other biomolecules, enabled their terminal oxygen and hydroxide groups to bind Ca and Se ions. The porous Se-(Ca(OH)2) nanocomposite showed high efficacy in interacting with seed embryos and stimulating germination  seeds using diphenylene iodonium chloride and uniconazole, as inhibitors of H2O2 and GA synthesis, respectively. The study evidenced that GA and H2O2 were essential for seed germination by decreasing ABA/GA ratio and stimulating reserve mobilization  was successfully applied to onion , mediated by IRES-specific trans-acting factors (ITAFs), is a key step that triggers m7G cap-independent translation of mRNAs investigated in the context of seed germination  seeds  has been recently used to assess changes in protein levels during seed germination .A key aspect of the pre-germinative metabolism is the molecular network underlying DNA damage sensing, signaling, and repair, namely the highly conserved DNA damage response (DDR), essential for genome maintenance  seeds  pathway in which DNA glycosylases remove oxidized bases, generating abasic sites that are subsequently filled through DNA synthesis. The most frequent oxidation product is 8-oxoguanine, removed by either the 8-oxoguanine DNA glycosylase/lyase (OGG1) or formamidopyrimidine-DNA glycosylase (FPG) , FPG1 (formamidopyrimidine-DNA glycosylase), and TDP1 (tyrosyl-DNA phosphodiesterase) associated with metabolic resumption has been reported in imbibed seeds of Medicago truncatula  acting on base damage/single-strand breaks (SSBs) and bulky lesions, as well as non-homologous end joining (NHEJ), alternative NHEJ pathways , and homologous repair (HR) involved in the repair of double-strand breaks (DSBs). Such mechanisms have been investigated in the context of pre-germinative metabolism to assess their impact on seed quality, their involvement in the response to priming, and their potential as sources of seed quality hallmarks Fig. . The higcis–trans isomerase, required for proper folding through proline isomerization, and heat shock proteins that protect against misfolding, are included in the set of relevant repair proteins active in germinating seeds , but a relevant issue is also the reduced shelf-life of the primed seeds , validation of 5.8S rRNA as a stress hallmark, at least in legumes, will necessitate testing across a wider range of species and/or genotypes.Among the parameters that should be kept under control, there is the duration of the rehydration step. When the treatment is prolonged, the resulting metabolic acceleration can easily bring seeds to overcome the critical threshold that leads to irreversible germination Fig. . When suHow to tackle inter- and intra-specific variability in assessing and improving seed quality? Pagano et al.  were able to improve seed storability , carbon dioxide (CO2), and/or pH  is widely used to measure not only DNA damage accumulation  but also the dynamics of DNA repair  useful to detect the superoxide radical  and oxidative damage (cytotoxic effect) makes their detection and quantification a relevant step in the assessment of seed quality, especially in the context of seed priming. ROS detection is challenging due to its low concentration and short half-life  caused by suboptimal treatments. Would it be possible to identify universal hallmarks of the seed response to priming useful or would be more reasonable to think about indicators restricted to specific taxonomic groups, e.g., legumes? To what extent storability of primed seeds can be improved? These are the challenges of seed priming, already taken up by the scientific community. Such challenges should be taken up and addressed, also considering the feasibility of seed priming at the field level. When dealing with fragile ecosystems, the choice of the most appropriate priming approach should be carefully handled to maximize the benefits. Participatory trials with local farmers should help seed technologists and researchers in the validation of candidate treatments.Given the current advances in seed priming and the efforts made to understand the molecular dynamics underlying either the benefits or the drawbacks of this technique, which recommendations should be given to the seed operators  of the private and public sectors? Is it possible to draw some guidelines? Although promising results need to be consolidated through the extensive validation of novel hallmarks, seed technologists should be more focused on finding out novel treatments, working on combinations of priming agents, and exploiting new types of materials.