Lymphoangiocrine signals promote cardiac growth and repair.

Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1-6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.

Can polarization of macrophage metabolism enhance cardiac regeneration?

While largely appreciated for their antimicrobial and repair functions, macrophages have emerged as indispensable for the development, homeostasis, and regeneration of tissue, including regeneration of the neonatal heart. Upon activation, mammalian neonatal macrophages express and secrete factors that coordinate angiogenesis, resolution of inflammation, and ultimately cardiomyocyte proliferation. This is contrary to adult macrophages in the adult heart, which are incapable of inducing significant levels of cardiac regeneration. The underlying mechanisms by which pro-regenerative macrophages are activated and regulated remain vague. A timely hypothesis is that macrophage metabolism contributes to this proliferative and regenerative potential. This is because we now appreciate the significant contributions of metabolites to immune cell programming and function, beyond solely bioenergetics. After birth, the metabolic milieu of the neonate is subject to significant alterations in oxygenation and nutrient supply, which will affect how metabolic substrates are catabolized. In this context, we discuss potential roles for select macrophage metabolic pathways during cardiac regeneration.

Identification and targeting of novel CDK9 complexes in acute myeloid leukemia.

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

Cardiopulmonary Bypass-Induced Inflammation and Myocardial Ischemia and Reperfusion Injury Stimulates Accumulation of Soluble MER.

OBJECTIVES: Soluble MER has emerged as a potential biomarker for delayed resolution of inflammation after myocardial injury and a therapeutic target to reduce cardiac-related morbidity and mortality in adults. The significance of soluble MER in pediatric populations, however, is unclear. We sought to investigate if soluble MER concentrations change in response to myocardial ischemia and reperfusion injury in pediatric patients. In parallel, we also sought to investigate for correlations between the change in soluble MER concentration and specific patient, bypass, and postoperative data. DESIGN: We quantified the change in plasma soluble MER concentration post- compared with precardiopulmonary bypass for each patient in a cohort of pediatric patients. Linear regression, correlation coefficients, and t tests were used to compare innate patient characteristics (i.e., sex, age, cyanotic vs acyanotic cardiac lesion), cardiac bypass data (i.e., total cardiac bypass time, total aortic cross-clamp time, perioperative steroid administration), and postcardiac bypass data (total postoperative ventilator days, total postoperative vasoactive medication days, and total postoperative ICU days) with change in soluble MER concentrations. SETTING: Whole blood samples were obtained intraoperatively at a single tertiary care children's hospital from April to October 2019. SUBJECTS: Our patient cohort included 24 pediatric patients ages ranging from birth to 19 years old with both cyanotic and acyanotic cardiac lesions. INTERVENTIONS: Retrospective analyses of pediatric blood specimens, as well as patient, bypass, and postoperative data, were performed. MEASUREMENTS AND MAIN RESULTS: We observed a statistically significant increase in soluble MER concentration post cardiac bypass in 17 of 24 patients (71%). CONCLUSIONS: Soluble MER concentrations increase with cardiopulmonary bypass-induced inflammation and myocardial ischemia and reperfusion injury in pediatric patients. The utility of soluble MER as a clinical biomarker to identify pediatric patients at risk for exacerbated postoperative outcomes after bypass-induced myocardial ischemia and reperfusion injury requires further investigation.

HIF-1α is Required to Differentiate the Neonatal Macrophage Secretome from Adults.

The immune response to stress diverges with age, with neonatal macrophages implicated in tissue regeneration versus tissue scarring and maladaptive inflammation in adults. Integral to the macrophage stress response is the recognition of hypoxia and pathogen-associated molecular patterns (PAMPs), which are often coupled. The age-specific, cell-intrinsic nature of this stress response remains vague. To uncover age-defined divergences in macrophage crosstalk potential after exposure to hypoxia and PAMPs, we interrogated the secreted proteomes of neonatal versus adult macrophages via non-biased mass spectrometry. Through this approach, we newly identified age-specific signatures in the secretomes of neonatal versus adult macrophages in response to hypoxia and the prototypical PAMP, lipopolysaccharide (LPS). Neonatal macrophages polarized to an anti-inflammatory, regenerative phenotype protective against apoptosis and oxidative stress, dependent on hypoxia inducible transcription factor-1α ( HIF-1α). In contrast, adult macrophages adopted a pro-inflammatory, glycolytic phenotypic signature consistent with pathogen killing. Taken together, these data uncover fundamental age and HIF-1α dependent macrophage programs that may be targeted to calibrate the innate immune response during stress and inflammation.

Metabolism: How removal of damaged cells impacts energy availability in the retina.

Once thought to be a quiescent process, elimination of damaged cells by professional phagocytes is now understood to modulate metabolite availability within tissues. A new study reveals that the retinal pigment epithelium serves as a local source of insulin after engulfment of damaged photoreceptors.

Macrophage-endothelial cell crosstalk orchestrates neutrophil recruitment in inflamed mucosa.

Neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires transendothelial migration (TEM). TEM involves several well-studied sequential adhesive interactions with vascular endothelial cells (ECs); however, what initiates or terminates this process is not well-understood. Here, we describe what we believe to be a new mechanism where vessel-associated macrophages through localized interactions primed EC responses to form ICAM-1 "hot spots" to support PMN TEM. Using real-time intravital microscopy of LPS-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we found that macrophage vessel association is critical for the initiation of PMN-EC adhesive interactions, PMN TEM, and subsequent accumulation in the intestinal mucosa. Anti-colony stimulating factor 1 receptor Ab-mediated macrophage depletion in the lamina propria and at the vessel wall resulted in elimination of ICAM-1 hot spots impeding PMN-EC interactions and TEM. Mechanistically, the use of human clinical specimens, TNF-α-KO macrophage chimeras, TNF-α/TNF receptor (TNF-α/TNFR) neutralization, and multicellular macrophage-EC-PMN cocultures revealed that macrophage-derived TNF-α and EC TNFR2 axis mediated this regulatory mechanism and was required for PMN TEM. As such, our findings identified clinically relevant mechanisms by which macrophages regulate PMN trafficking in inflamed mucosa.

Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice.

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation.

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Hypoxia-inducible factors individually facilitate inflammatory myeloid metabolism and inefficient cardiac repair.

Hypoxia-inducible factors (HIFs) are activated in parenchymal cells in response to low oxygen and as such have been proposed as therapeutic targets during hypoxic insult, including myocardial infarction (MI). HIFs are also activated within macrophages, which orchestrate the tissue repair response. Although isoform-specific therapeutics are in development for cardiac ischemic injury, surprisingly, the unique role of myeloid HIFs, and particularly HIF-2α, is unknown. Using a murine model of myocardial infarction and mice with conditional genetic loss and gain of function, we uncovered unique proinflammatory roles for myeloid cell expression of HIF-1α and HIF-2α during MI. We found that HIF-2α suppressed anti-inflammatory macrophage mitochondrial metabolism, while HIF-1α promoted cleavage of cardioprotective MerTK through glycolytic reprogramming of macrophages. Unexpectedly, combinatorial loss of both myeloid HIF-1α and HIF-2α was catastrophic and led to macrophage necroptosis, impaired fibrogenesis, and cardiac rupture. These findings support a strategy for selective inhibition of macrophage HIF isoforms and promotion of anti-inflammatory mitochondrial metabolism during ischemic tissue repair.

Select Macrophage Noncoding RNAs of Interest in Cardiovascular Disease.

Cardiovascular disease remains a leading cause of morbidity and mortality worldwide. Aspects of disease severity that are associated with heightened inflammation, such as during atherosclerosis or after myocardial infarction, are correlated with macrophage activation and macrophage polarization of the transcriptome and secretome. In this setting, non-coding RNAs (ncRNAs) may be as abundant as protein-coding genes and are increasingly recognized as significant modulators of macrophage gene expression and cytokine secretion, although the functions of most ncRNAs-and in particular, long non-coding RNAs-remain unknown. Herein, we discuss a subset of specific ncRNAs of interest in macrophages in atherosclerosis and during myocardial inflammation.

Single-cell RNA sequencing uncovers heterogenous transcriptional signatures in macrophages during efferocytosis.

Efferocytosis triggers cellular reprogramming, including the induction of mRNA transcripts which encode anti-inflammatory cytokines that promote inflammation resolution. Our current understanding of this transcriptional response is largely informed from analysis of bulk phagocyte populations; however, this precludes the resolution of heterogeneity between individual macrophages and macrophage subsets. Moreover, phagocytes may contain so called "passenger" transcripts that originate from engulfed apoptotic bodies, thus obscuring the true transcriptional reprogramming of the phagocyte. To define the transcriptional diversity during efferocytosis, we utilized single-cell mRNA sequencing after co-cultivating macrophages with apoptotic cells. Importantly, transcriptomic analyses were performed after validating the disappearance of apoptotic cell-derived RNA sequences. Our findings reveal new heterogeneity of the efferocytic response at a single-cell resolution, particularly evident between F4/80+ MHCIILO and F4/80- MHCIIHI macrophage sub-populations. After exposure to apoptotic cells, the F4/80+ MHCIILO subset significantly induced pathways associated with tissue and cellular homeostasis, while the F4/80- MHCIIHI subset downregulated these putative signaling axes. Ablation of a canonical efferocytosis receptor, MerTK, blunted efferocytic signatures and led to the escalation of cell death-associated transcriptional signatures in F4/80+ MHCIILO macrophages. Taken together, our results newly elucidate the heterogenous transcriptional response of single-cell peritoneal macrophages after exposure to apoptotic cells.

Immunometabolism of Phagocytes and Relationships to Cardiac Repair.

Cardiovascular disease remains the leading cause of death worldwide. Myocardial ischemia is a major contributor to cardiovascular morbidity and mortality. In the case of acute myocardial infarction, subsequent cardiac repair relies upon the acute, and coordinated response to injury by innate myeloid phagocytes. This includes neutrophils, monocytes, macrophage subsets, and immature dendritic cells. Phagocytes function to remove necrotic cardiomyocytes, apoptotic inflammatory cells, and to remodel extracellular matrix. These innate immune cells also secrete cytokines and growth factors that promote tissue replacement through fibrosis and angiogenesis. Within the injured myocardium, macrophages polarize from pro-inflammatory to inflammation-resolving phenotypes. At the core of this functional plasticity is cellular metabolism, which has gained an appreciation for its integration with phagocyte function and remodeling of the transcriptional and epigenetic landscape. Immunometabolic rewiring is particularly relevant after ischemia and clinical reperfusion given the rapidly changing oxygen and metabolic milieu. Hypoxia reduces mitochondrial oxidative phosphorylation and leads to increased reliance on glycolysis, which can support biosynthesis of pro-inflammatory cytokines. Reoxygenation is permissive for shifts back to mitochondrial metabolism and fatty acid oxidation and this is ultimately linked to pro-reparative macrophage polarization. Improved understanding of mechanisms that regulate metabolic adaptations holds the potential to identify new metabolite targets and strategies to reduce cardiac damage through nutrient signaling.