HomeCirculation ResearchVol. 119, No. 1Circulation Research “In This Issue” Anthology Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessResearch ArticlePDF/EPUBCirculation Research “In This Issue” Anthology Ruth Williams and and The Editors Ruth WilliamsRuth Williams Search for more papers by this author and and The Editors Search for more papers by this author Originally published24 Jun 2016https://doi.org/10.1161/RES.0000000000000108Circulation Research. 2016;119:e1–e27Pik3cb Links Hippo-YAP and PI3K-AKT Pathways (p 35)1Lin et al reveal how Yes-associated protein activates cardiomyocyte proliferation.Download figureDownload PowerPointSignaling via the Hippo kinase cascade results in the nuclear localization and activation of transcriptional co-activator YAP (Yes-associated protein). The Hippo-YAP pathway promotes cardiomyocyte proliferation in the developing fetal heart and, when over-expressed, in adult cardiomyocytes as well. The transcriptional targets of YAP that control proliferation, however, remain largely unknown. To identify the targets of this pathway, Lin and colleagues performed genome-wide chromatin immunoprecipitation experiments with YAP as well as genome-wide transcriptional profiling of cells that had a gain or loss of function of YAP. The results obtained from these experiments revealed a total of 26 genes that bound directly to YAP (together with its DNA-binding partner, TEAD), and showed increased or reduced transcriptional activity. Among the genes that were upregulated was pik3cb, a gene that encodes part of the catalytic subunit of the kinase PIK3, which has been found in other independent studies to drive cardiomyocyte proliferation. Lin and colleagues went on to show that YAP-induced proliferation required functional pik3cb, and that heart-specific depletion of YAP, which causes hypertrophy and heart failure in mice, could be rescued in part by boosting the expression of pik3cb. Taken together these results indicate that increasing YAP or pik3cb expression could be a useful strategy to stimulate cardiomyocyte proliferation and thereby promote myocardial regeneration after injury.miR-208 in Right Ventricular Failure (p 56)2Paulin et al identify a right ventricle-specific mechanism in pulmonary hypertension and, with it, potential biomarkers of disease progression.Download figureDownload PowerPointUnlike systemic hypertension, which leads to left ventricular failure, pulmonary hypertension results in right ventricular failure (RVF). And even though in both conditions cardiac hypertrophy occurs as a compensatory mechanism, in RVF this brief compensation is quickly followed by a catastrophic decompensation phase and increased mortality. To investigate how RVF transitions from compensation to decompensation, Paulin and colleagues studied a rat model of pulmonary hypertension. They analyzed RVF tissue during compensation and decompensation and discovered that the latter was associated with increased inflammation and decreased expression of a right ventricle-specific transcription factor called Mef2. Upon performing an unbiased microarray screen, they found that a number of microRNAs (miRs) were misregulated during decompensation. Among the down-regulated miRs was miR208, which normally suppresses expression of a transcription factor called MED, a transcription factor that itself suppresses Mef2. Thus the downregulation of miR208 in decompensation could increase MED, which in turn could further suppress the already diminished Mef2. Exactly how these factors contribute to RVF remains to be determined, but in the meantime these changes could serve as valuable biomarkers of disease progression.α-Catenins Regulate Yap Activity in the Heart (p 70)3Alpha-catenins suppress YAP and reduce proliferation in cardiomyocytes, report Li et al.Download figureDownload PowerPointLike Lin and colleagues, Li and colleagues have been studying the control of cardiomyocyte proliferation by the transcriptional co-activator YAP. But whereas Lin and colleagues searched for downstream targets of YAP, Li and colleagues have identified α-catenins as upstream regulators. Inside cells, α-catenin proteins link the junction proteins, cadherins, to the cytoskeleton, but aside from this structural role, α-catenins have been found, in some cell types, to control proliferation. It is thought that, through their interaction with the cadherin junctions, the α-catenins sense an increase in cell density and shut down cell division. Li and colleagues now show that this anti-proliferative effect also occurs in the heart. Specifically, they found that mouse hearts lacking the two types of α-catenin exhibited an approximately 23% increase in cardiomyocyte number. They also found that phosphorylation of YAP was reduced in the catenin-deficient hearts, which prevented YAP from entering the nucleus and activating proliferation genes. The team went on to show that compared with their wild-type counterparts, the mice lacking α-catenin displayed improved repair of heart tissue following myocardial infarction. The discovery lends further support to notion that the activation of YAP could be a potential regenerative therapyiCM Reprogramming Factor Stoichiometry (p 237)4Wang et al shuffle the gene order in reprogramming vectors and improve fibroblast-to-cardiomyocyte conversion.Download figureDownload PowerPointScar tissue that forms after myocardial infarction reduces heart function and can ultimately lead to heart failure and death. Fibroblasts of the scar tissue, however, can be directly reprogramed into cardiomyocytes—by transfecting the cells with the transcription factors Gata4 (G), Mef2 (M), and Tbx5 (T). While the approach holds great promise for regenerating functional heart muscle its clinical utility is limited by its inefficiency. Getting a precise balance of the three factors might be important for reprogramming efficiency. Thus, Wang et al made six polycistronic vectors—each with the factors in a different possible order (GMT, GTM, MGT and so on)—and compared their reprogramming capacities. They also compared these polycistronic vectors with combined yet separate vectors encoding G, M, and T. Transfection experiments with different vectors showed that each of the constructs produced different ratios of factor expression, and importantly different reprogramming efficiencies. The two constructs in which Mef2 was most 5’—and which happened to have the highest Mef2 expression—reprogrammed fibroblasts more efficiently than other vectors. Indeed, the MGT vector improved reprogramming 10-fold over that seen with individual factor vectors. This improved vector could serve as a platform for further mechanistic studies and optimization, say the authors.Cardiac Exosome Modeling and Therapy (p 255)5Gray et al discover that exosomes from hypoxia-treated cardiac progenitor cells promote heart regeneration.Download figureDownload PowerPointTo regenerate heart tissue after a myocardial infarction, researchers have investigated cell therapies involving cardiac progenitor cells. These cells can differentiate into heart cells such as cardiac myocytes, endothelial cells and fibroblasts, but it is not clear whether this differentiation is the sole or even main reason for the improvement of cardiac function after transplantation with progenitor cells. Indeed, there is evidence to support the notion that the paracrine effects of factors secreted by these cells contribute to their beneficial effects. In support of this idea, it has been shown that many cell types, including cardiac progenitors, secrete membrane-bound vesicles called exosomes that can exert certain paracrine effects. Gray and colleagues therefore studied the effects of cardiac progenitor-derived exomes on cardiac cells in vitro and in vivo. To mimic infarction they exposed the progenitors to hypoxia before collecting the exosomes. They showed that compared with exosomes derived under normoxic conditions these hypoxic exosomes induced capillary-like tube growth in cardiac endothelial cells and reduced expression of fibrosis-associated genes in fibroblasts. Moreover these exosomes improved the function of infarcted rat hearts. The team went on to identify specific microRNAs contained in the exosomes that were associated with hypoxia, which could have positive effects on cardiac function and therefore warrant further analysis.CD34+ Cells and Mortality (p 289)6Low numbers of circulating progenitor cells indicate high risk of death in coronary artery disease patients, report Patel et al.Download figureDownload PowerPointProgenitor cells, originating primarily from the bone marrow, are important for the regeneration and repair of injured blood vessels. Patel and colleagues therefore hypothesized that a lack of progenitor cells in a person’s blood might indicate an impaired ability for vessel regeneration. To test their hypothesis, the team recruited two cohorts of 502 and 403 patients with coronary artery disease. They counted the progenitor cell numbers in the patients’ blood, and followed their progress for 2.7 years and 1.2 years respectively. The team defined progenitor cells as mononuclear cells expressing the marker CD34. They also examined subsets of these cells that expressed CD133 (a marker of more primitive stem cells), VEGFR2, or CXCR4 (both associated with stem cell recruitment and homing). The team found in both cohorts that the number of CD34+− or CD34+/CD133− cells in a person’s blood was inversely correlated with risk of all-cause death, as well as cardiovascular mortality. Indeed patients with the lowest number of these progenitor cells were almost three times more likely to die. These results suggest not only that progenitor cells may be a useful biomarker for coronary artery disease prognosis, but also that these cells may be useful targets for regenerative therapies.Stroke Activates the Bone Marrow (p 407)7Courties et al identify the source of inflammatory immune cells after a stroke.Download figureDownload PowerPointStroke is caused by an interruption of blood flow to the brain due to hemorrhage or occlusion of a blood vessel. The resulting ischemic injury triggers the recruitment of inflammatory immune cells, which are essential for clearing the damaged tissue, but can cause further damage if the response is excessive or prolonged. To determine whether immune cells were recruited to the brain from elsewhere in the body after a stroke, or were made afresh in the bone marrow, Courties and colleagues studied the bone marrow cell activity in mice after experimentally induced stroke. They found that rates of cell division—specifically of hematopoietic stem cells (HSC)—were increased. As a result, the numbers of both neutrophils and monocytes were increased as well. There was also an increase in the bone marrow levels of noradrenaline, and the team found that this hormone drove the HSC proliferation: mice that lacked the β3 adrenergic receptor for noradrenaline failed to exhibit increased HSC proliferation after stroke. Knowing the source of the immune cells may offer new ways to reduce their recruitment to the brain and thus diminish tissue damage after a stroke. But given the essential role of the immune response in tissue repair, further studies will be necessary to determine how to fine tune the response to maximize brain recovery.MicroRNA Induced Cardiac Reprogramming In Vivo (p 418)8In vivo cardiac reprogramming with microRNAs improves heart function, report Jayawardena et al.Download figureDownload PowerPointThe scar tissue formed after a myocardial infarction reduces the contractility and function of the heart; leading often to heart failure. To simultaneously reduce scar tissue and increase heart function researchers are investigating ways to reprogram the scar fibroblasts directly into cardiomyocytes. Such reprogramming has been shown to work in mice using different combinations of either transcription factors or microRNAs (miRs). But while reprogramming with transcription factors has been shown to result in functional improvements in the heart muscle, whether the same is true for miR-based reprogramming is not known. Moreover, it is unclear whether miR reprogramming leads to the formation of fully differentiated cardiac myocytes. Jayawardena and colleagues therefore examined miR-based reprogramming more closely. They found that around the injury zone of infarcted mouse hearts injection with the miRs led to a three-fold increase in cells expressing a marker of mature cardiac myocytes. Furthermore, these reprogrammed cells closely resembled mature ventricular cardiac myocytes in their electrophysiological behavior. But importantly, the team showed that miR reprogramming of injured hearts improved heart function in mice. Although reprogramming with either method—transcription factors or miRs—remains inefficient, these new results validate the miR-mediated reprogramming as a viable method worthy of further optimization.TMAO Promotes Renal Fibrosis and Dysfunction (p 448)9A choline-rich diet may contribute to chronic kidney disease, say Tang et al.Download figureDownload PowerPointDuring the breakdown of dietary choline, microbes residing in the gut generate the metabolite trimethylamine N-oxide (TMAO). High levels of TMAO in the blood have been associated with both chronic kidney disease (CKD) and coronary artery disease, but it is not clear whether TMAO can directly contribute to these conditions. Hence, Tang and colleagues examined the link between TMAO and CKD in a large cohort of subjects and discovered that individuals with CKD had markedly higher TMAO levels than those without CKD. Furthermore, within the group of CKD patients, those with the highest levels of TMAO were at a greater risk of dying. To determine whether TMAO could directly lead to CKD, the team fed mice a diet rich in either TMAO, or choline. They found that when placed for six weeks on either diet, the mice displayed increased blood levels of TMAO. The mice also exhibited signs of renal injury, characterized by increased amounts of the kidney injury marker 1 (KIM1), fibrosis of the kidney tubule interstitium, and impaired renal function. Together the results suggest not only that TMAO is a useful prognostic marker of CKD, but that diets low in choline—which is found in red meat, egg yolks, liver, and high-fat dairy products—may help in preventing, or slowing the progression of, CKD.CAESAR: A New Paradigm to Study Cardioprotection (p 572)10Jones et al bring clinical-level rigor to preclinical assessments of cardioprotective interventions.Download figureDownload PowerPointDespite forty years of effort and considerable financial investment, the cardioprotection field has yet to develop a single drug capable of reducing infarct size in heart attack patients. This failure in translating preclinical studies to the clinic has been ascribed in part to the lack of reproducibility and rigor of preclinical studies. To address this problem, the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR) was established with the aim of ensuring consistent methods across multiple centers and improving accuracy by adopting approaches such as randomization of animal subjects and blinding of investigators to treatment groups. To evaluate the efficacy of this approach, the consortium performed experiments in which the hearts were preconditioned by a mild ischemia to protect against subsequent infarction. Mice, rabbits and pigs were treated at a minimum of two different centers per species and protocols were modified to ensure that the treatment of animals, performance of experiments and the measurements of resulting infarct sizes remained consistent across each site. Using this new approach, the investigators found that the protection provided by preconditioning and the reduction in infarct sizes were closely comparable between the different centers. These results signal the launch of operational CAESAR protocols—now freely available to any researcher—and the start of a new more rigorous era in the field of cardioprotective drug discovery.Circulating Cells Contribute to Myocardial Repair (p 633)11Wu et al confirm bone marrow cells can promote cardiac regeneration in vivo.Download figureDownload PowerPointThe adult heart has only a limited capacity for regeneration. Therefore, identifying specific cells that contribute and could be boosted to improve recovery from injury could lead to significant advances in the field. It is widely accepted, for example, that bone marrow stem cells can differentiate into cardiomyocytes, but it is unclear whether they actually contribute to regeneration in vivo. Indeed, bone marrow transfer experiments have shown that donor cells can home to an injured heart, engraft, and express cardiomyocyte markers. But parabiosis experiments, where the circulatory systems of two mice—one with labeled cells and one wildtype—are joined, have shown only the engraftment of cells, with no sign of differentiation into cardio myocytes. Wu and colleagues wondered whether the timing of parabiosis—which in previous experiments was performed immediately prior to heart injury—might be a crucial factor. They found that in parabiotically-paired mice, it takes 7 to 10 days to establish a fully shared circulatory system. Informed by these findings, the team delayed heart injury until that time and observed that cells from the labeled mouse not only integrated into the injured heart of the unlabeled mouse, but also developed into cardio myocytes. They suggest that in prior experiments, early injury signals failed to pass through the shared circulation, which was only partially complete. Importantly, these findings support the result of bone marrow transfer experiments and suggest bone marrow cells are valuable targets for boosting heart regeneration.Cell Therapy for Hypoplastic Left Heart Syndrome (p 653)12Ishigami et al perform the first clinical trial for cell therapy in hypoplastic left heart syndrome.Download figureDownload PowerPointHypoplastic left heart syndrome (HLHS) is a congenital heart malformation caused by the incomplete development of the left ventricle, aorta and valves. Infants with HLHS are unable to properly pump blood through the body and therefore require immediate surgical intervention—either to adapt the patient’s own heart, or to replace it with a transplanted one. The former approach is unable to fully restore circulation and further surgeries are generally required, while the latter approach depends upon an all-too-scarce donor. Thus additional therapeutic approaches are needed for the treatment of HLHS. Ishigami and colleagues investigated the possibility of progenitor cell therapy as one such approach. Recent research has shown that in HLHS patients, cardiac myocyte proliferation declines during development—a problem that might be resolved by the transfer of progenitor cells. Moreover, initial clinical trials in myocardial infarction patients indicate that progenitor cell therapy thickens cardiac muscle. The team thus isolated and expanded progenitor cells from seven HLHS patients and then transferred the cells back into the patients’ hearts by intracoronary infusion one month after palliative surgery. Eighteen months later, patients exhibited improved right ventricle function and reduced heart failure status, suggesting that the approach warrants additional clinical trials.Macrophage Sortilin Promotes LDL Uptake, Foam Cell Formation, and Atherosclerosis (p 789)13Sortilin protein drives cholesterol uptake in macrophages and promotes atherosclerosis, report Patel et al.Download figureDownload PowerPointA characteristic feature of atherosclerosis is the formation of foam cells that arise from cholesterol-loaded macrophages. But exactly how macrophages take up cholesterol from transporter proteins such as low-density lipoprotein (LDL) is unclear, especially since the deletion of known macrophage lipoprotein receptors in mice does not prevent foam cell formation. Patel and colleagues thus turned their attention to a protein called sortilin. This protein mediates hepatic LDL uptake and, through genome-wide association studies, has been linked with coronary artery disease. The function of sortilin in macrophages, however, was unknown. The team found that atherosclerosis-prone mice lacking sortilin—either in all cells or just macrophages—showed a significant reduction in the number of atherolsclerotic lesions compared to mice with normal levels of the protein. They also discovered that sortilin-lacking macrophages exhibited a reduction in LDL uptake, while macrophages that over-expressed sortilin actually increased their LDL uptake. These results suggest that preventing the sortilin-driven uptake of LDL in macrophages might reduce foam cell formation and thus be a novel strategy for treating, and perhaps even preventing, atherosclerosis in the future.Transcriptional Reversion of Cardiomyocyte Fate During Mammalian Cardiac Regeneration (p 804)14O’Meara et al describe the transcriptional signature of mammalian heart regeneration.Download figureDownload PowerPointThe adult mammalian heart has a limited capacity for regeneration. In contrast, neonatal mice, within the first week of life, can completely repair their hearts after injury. During this regeneration, neonatal heart cells lose their sarcomeric structures, adopt a less-differentiated state, and reenter the cell cycle. If this same regenerative process could be recapitulated in the adult hearts, full recovery from myocardial injury might be possible. Hence to understand the mechanism underlying the regenerative capacity of the neonatal heart, O’Meara and colleagues examined changes in the global gene expression profiles of the neonatal heart after injury and during regeneration. They also studied the expression profiles of normal neonatal and adult cardiomyocytes, mouse embryonic stem cells during differentiation into cardiomyocytes, and adult cardiomyocytes induced to de-differentiate. After cross-referencing these data, the team identified a reference panel of genes involved in the differentiation and regeneration of a normal heart. They found that injury to the neonatal heart essentially reversed the differentiation process such that seven days post-injury the genes encoding sarcomeric proteins were reduced, while the expression of cell cycle genes was elevated. The team highlighted IL-13, STAT3 and STAT6 as regulators of cell cycle re-entry, but their results provide a large number of additional candidates that could be examined in future regeneration studies.Effects of DNA Damage in Smooth Muscle Cells in Atherosclerosis (p 816)15DNA damage promotes atherosclerotic plaque instability report Gray et al.Download figureDownload PowerPointDNA damage occurs in the vascular smooth muscle cells (VSMCs) of atherosclerotic plaques and it becomes more pervasive with disease progression. But whether such damage is a cause or consequence of the condition is unclear. It is known that DNA damage reduces cell proliferation—because cells stop cycling to repair the DNA, and this could arguably slow down plaque growth. If, however, the damage is severe, senescence and apoptosis can occur, which induces proinflammatory cytokine production and immune cell recruitment, thus exacerbating plaque growth. To better define the relationship between plaque growth and DNA damage, Gray and colleagues studied the effects of accelerating or inhibiting DNA repair in human VSMC from normal aorta and atherosclerotic plaques. As expected, they found that VSMCs from plaques had significantly higher levels of DNA damage as well as increased activation of DNA repair pathways. By accelerating this repair in a mouse model of atherosclerosis, they found that while plaque size remained the same, there was an increase in the stability of the fibrous caps of the plaques. Inhibiting repair, on the other hand, promoted plaque instability. Because unstable plaques are associated with a greater risk of myocardial infarction and stroke, preventing DNA damage, or promoting DNA repair could be a novel therapeutic strategy for enhancing plaque stability in patients with atherosclerotic disease.Small HDL Particles Export Cholesterol via ABCA1 (p 1133)16High density lipoproteins (HDLs) may be “good” cholesterols, but Du et al discover which are the best of the best.Download figureDownload PowerPointCirculating HDLs collect cholesterol exported from cells and ferry it to the liver for excretion in the bile. Because of this property high HDL levels are believed to protect against cardiovascular disease. However, HDLs are a heterogeneous population of particles and it is not clear whether some are more efficient transporters than others. Moreover, the relative efficiency of the cellular exporters, ABCA1 and ABCG1, which transfer cholesterol from inside cells to the awaiting transporter, has not been determined. Hence, Du and colleagues examined the cholesterol transporting efficiency of different HDL subfractions and they found that the smallest particles, HDL3b and 3c, along with un-loaded apoA1—the main protein component of HDL—are much more efficient transporters of cholesterol from cells in culture than the large HDL particles. They also found the more efficient HDL particles export cholesterol via ABCA1 rather than ABCG1. Together the results suggest that cardioprotective therapies aimed at reducing tissue cholesterol levels might work best if they specifically boost small HDLs and the activity of ABCA1.lncRNA-MIAT and Microvascular Dysfunction (p 1143)17Yan et al identify the long non- coding RNA MIAT as a proangiogenic factor in diabetic retinopathy.Download figureDownload PowerPointAngiogenesis is essential for tissue growth, development, and wound healing, but it can also be detrimental in diseases such as cancer or diabetic retinopathy. A better understanding of the processes that regulate angiogenesis may therefore reveal ways to control it in a variety of clinical scenarios. To search for novel angiogenic factors that regulate pathological retinal angiogenesis associated with diabetes, Yan and colleagues focused on long, noncoding RNAs (lncRNAs)—RNAs of 200 nucleotides or more that regulate gene expression. They found that MIAT (myocardial infarction associated transcript) is upregulated in the retinas of patients and rats with diabetes as well as in endothelial cells treated with high levels of glucose. The proangiogenic factor VEGF was also increased in the diabetic rat retinas, and the team showed that knocking down MIAT reduced retinal VEGF expression, improved vascular abnormalites as well as visual function. The team also provides evidence to suggest that MIAT promotes VEGF expression by binding and inhibiting a microRNA that normally suppresses VEGF. The finding that MIAT inhibition can improve diabetes-induced retinal microvascular abnormalities suggests this lncRNA could be a target for treating retinopathy, which remains one of the most significant secondary complications of advanced diabetes.Coronary NET Burden and DNase Activity in STE-ACS (p 1182)18Mangold et al discover that higher levels of neutrophil NETs in a coronary thrombus are associated with more severe infarction.Download figureDownload PowerPointFormation of a large thrombus after acute plaque rupture leads to poor tissue perfusion and is associated with high morbidity and mortality Researchers are therefore investigating ways to rapidly breakdown such thrombi to re-establish blood flow in affected patients. Mangold and colleagues now suggest that one method could be to eliminate chromatin NETs—or neutrophil extracellular traps—from the thrombi. Neutrophils release their own nuclear DNA and proteins (chromatin) in the form of NETs to ensnare pathogens and stimulate proinflammatory responses—in much the same way Spiderman catches villains in his web. However the formation of these sticky, stringy traps can be problematic if they coincide with thrombus formation. By analyzing thrombi from 111 male patients with acute coronary syndrome, Mangold and colleagues found that the abundance of NETs in thrombi was positively correlated with infarct size. Encouragingly, however, the patients’ DNase level negatively correlated with both the abundance of NETs and infarct size. That is, patients with more DNase had smaller infarcts. DNase digests chromatin and the team found that the enzyme accelerated degradation of thrombi in vitro. Thus, boosting DNase activity at the site of thrombi might be a way to speed their removal.Flecainide and RyR2 (p 1324)19The anti-arrhythmia drug flecainide does not block ryanodine receptors, report Bannister et al.Download figureDownload PowerPointPatients who suffer catecholaminergic polymorphic ventricular tachycardia (CPVT) exhibit dysfunctional calcium release from cardiomyocyte sarcoplasmic reticulum (SR) during times of increased adrenaline—such as during exercise or emotional stress. β-adrenergic receptor blockers are generally prescribed to minimize cardiac excitability, but for some patients the tachycardia remains poorly controlled. The drug flecainide can be given to such patients. Exactly how flecainide works in CPVT, however, is a matter of controversy. Some researchers suggest that, in addition to its known sodium channel-blocking ability, flecainide blocks ryanodine receptors (RyR2)—the calcium ion channels in SR. Others argue, however, that flecainide only blocks ion flow through RyR2 in a non-physiological direction: from cytoplasm into SR. To settle the issue, Bannister and colleagues studied ion flow through recombinant human RyR2 channels in phospholipid bilayers. They found t