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The field of cardio-oncology has emerged in response to the increased risk of cardiovascular disease (CVD) in patients with cancer. However, recent studies suggest a more complicated CVD-cancer relationship, wherein development of CVD, either prior to or following a cancer diagnosis, can also lead to increased risk of cancer and worse outcomes for patients. In this review, we describe the current evidence base, across epidemiological as well as preclinical studies, which supports the emerging concept of ‘reverse-cardio oncology’, or CVD-induced acceleration of cancer pathogenesis.

BACKGROUND:Prediction of severity of Post-COVID-19 condition is not clear. We determined whether hospital course variables correlated with objective and subjective outcomes 3- and 6-months post-COVID-19 onset.METHODS:Adults admitted to hospital with acute COVID-19 were recruited (Feb 15, 2020-April 1, 2021) from two tertiary hospitals in Vancouver, Canada. We recorded baseline characteristics, COVID severity score, organ function, ICU and hospital length of stay. Survivors were evaluated at 3- and 6-months post- COVID-19 onset: subjective: University of California San Diego Shortness of Breath Score, EuroQol 5D Visual Analogue Scale, Frailty Index; Objective: functional capacity (6-minute walk test), pulmonary (pulmonary function tests), cardiac (echocardiography, BNP), renal (creatinine), hepatic (AST, ALT, bilirubin), and coagulation (aPTT, INR, D-dimers, fibrinogen).RESULTS:In 133 post-COVID-19 patients (age 62 ± 14 years, 65% male with frequent comorbidities [hypertension (42%), diabetes (29%), chronic cardiac (22%)), 42% were admitted to Intensive Care Unit: 23%, 23% and 2% required ventilation, vasopressors or renal support, respectively. One-third had restrictive lung function (CONCLUSIONS:Early organ dysfunction predicts later objective pathology especially restrictive lung disease and a pro-coagulant state. These hypothesis-generating findings require further validation in additional patients.

Unhealthful lifestyle factors, such as obesity, disrupt organismal homeostasis and accelerate cancer pathogenesis, partly through metabolic and immunological dysregulation. Exercise is a prototypical strategy that maintains and restores homeostasis at the organismal, tissue, cellular and molecular levels and can prevent or inhibit numerous disease conditions, including cancer. Here, we review unhealthful lifestyle factors that contribute to metabolic and immunological dysregulation and drive tumourigenesis, focusing on patient physiology (host)-tissue-tumour microenvironment interactions. We also discuss how exercise may influence distant tissue microenvironments, thereby improving tissue function through both metabolic and immunospecific pathways. Finally, we consider future directions that merit consideration in basic and clinical translational exercise studies.

Dysregulation of cholesterol homeostasis is associated with many diseases such as cardiovascular disease and cancer. Liver X receptors (LXRs) are major upstream regulators of cholesterol homeostasis and are activated by endogenous cholesterol metabolites such as 27-hydroxycholesterol (27HC). LXRs and various LXR ligands such as 27HC have been described to influence several extra-hepatic biological systems. However, disparate reports of LXR function have emerged, especially with respect to immunology and cancer biology. This would suggest that, similar to steroid nuclear receptors, the LXRs can be selectively modulated by different ligands. Here, we use RNA-sequencing of macrophages and single-cell RNA-sequencing of immune cells from metastasis-bearing murine lungs to provide evidence that LXR satisfies the 2 principles of selective nuclear receptor modulation: (1) different LXR ligands result in overlapping but distinct gene expression profiles within the same cell type, and (2) the same LXR ligands differentially regulate gene expression in a highly context-specific manner, depending on the cell or tissue type. The concept that the LXRs can be selectively modulated provides the foundation for developing precision pharmacology LXR ligands that are tailored to promote those activities that are desirable (proimmune), but at the same time minimizing harmful side effects (such as elevated triglyceride levels).

Psychological stress is associated with elevated circulating markers of inflammation, including inflammatory cytokines and acute phase reactants that accelerate progression of chronic inflammatory diseases (e.g., atherosclerosis, metabolic syndrome, autoimmune diseases and cancer). However, the mechanisms underlying inflammatory reactivity to stress and how it confers future health risk are poorly understood. Monocyte-derived macrophages play key roles in sustaining tissue inflammation, and recent studies show that they can maintain epigenetic memory of pathogen or sterile inflammatory insults, leading to a heightened inflammatory state upon secondary stimulation. We show herein that psychological stress induces transcriptomic and epigenomic reprogramming of monocytes which primes them for a heightened inflammatory immune response following challenge. Monocytes isolated from stressed mice or humans with high reported levels of psychological stress exhibit a common signature of heightened inflammatory gene expression and produce increased levels of proinflammatory cytokines upon ex vivo stimulation with Toll-like receptor ligands. RNA and ATAC sequencing studies revealed that monocytes from stressed mice and humans exhibit activation of cellular metabolic pathways, including mTOR and PI3Kinase pathways, and reduced chromatin accessibility at loci associated with mitochondrial respiration. Taken together, our findings suggest that psychological stress primes the reprogramming of innate immune cells resulting in a hyperresponsive inflammatory state, which may explain its deleterious association with inflammatory disease risk.

Rationale:Therapeutic efforts to decrease atherosclerotic cardiovascular disease risk have focused largely on reducing atherogenic lipoproteins, yet lipid-lowering therapies alone are insufficient to fully regress plaque burden. We postulate that arterial repair requires resolution of a maladaptive immune response and that targeting factors that hinder inflammation resolution will facilitate plaque regression.Objective:The guidance molecule Ntn1 (netrin-1) is secreted by macrophages in atherosclerotic plaques, where it sustains inflammation by enhancing macrophage survival and blocking macrophage emigration. We tested whether silencing Ntn1 in advanced atherosclerosis could resolve arterial inflammation and regress plaques.Methods and Results:To temporally silenceNtn1in myeloid cells, we generated genetically modified mice in whichNtn1could be selectively deleted in monocytes and macrophages using a tamoxifen-induced CX3CR1-driven cre recombinase (Ntn1fl/flCx3cr1creERT2+) and littermate control mice (Ntn1fl/flCx3cr1WT). Mice were fed Western diet in the setting of hepatic PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression to render them atherosclerotic and then treated with tamoxifen to initiate deletion of myeloidNtn1(MøΔNtn1) or not in controls (MøWT). Morphometric analyses performed 4 weeks later showed that myeloid Ntn1 silencing reduced plaque burden in the aorta (−50%) and plaque complexity in the aortic root. Monocyte-macrophage tracing experiments revealed lower monocyte recruitment, macrophage retention, and proliferation in MøΔNtn1compared with MøWTplaques, indicating a restructuring of monocyte-macrophage dynamics in the artery wall upon Ntn1 silencing. Single-cell RNA sequencing of aortic immune cells before and afterNtn1silencing revealed upregulation of gene pathways involved in macrophage phagocytosis and migration, including theCcr7chemokine receptor signaling pathway required for macrophage emigration from plaques and atherosclerosis regression. Additionally, plaques from MøΔNtn1mice showed hallmarks of inflammation resolution, including higher levels of proresolving macrophages, IL (interleukin)-10, and efferocytosis, as compared to plaques from MøWTmice.Conclusion:Our data show that targeting Ntn1 in advanced atherosclerosis ameliorates atherosclerotic inflammation and promotes plaque regression.

Acute myocardial infarction rapidly increases blood neutrophils (2 hours). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (30 minutes) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues.Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilised splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model.Our findings show a novel EV-dependent mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.Peripheral blood neutrophils are rapidly elevated following acute myocardial infarction (AMI) and prior to alterations in systemic cytokines. Extracellular vesicles (EVs) are membrane enclosed particles that carry protein and miRNAs and are rapidly liberated from endothelial cells (EC). Here, we show that following AMI EC-derived-EVs (EC-EVs) mediate neutrophil mobilisation from the spleen via EC-EV-VCAM-1 and induce transcriptional activation of neutrophils in the blood to favour miRNA-126-mRNA targets; miRNA-126 antagomir treatment lowers infarct size. EC-EV-VCAM-1 and EC-EV-miRNA-126 are novel mechanisms that mobilise splenic reserve of neutrophils, a previously unidentified source of neutrophils in sterile ischaemic injury.

Rationale: MicroRNA-33 (miR-33 [post-transcriptionally in the rationale]) post-transcriptionally represses genes involved in lipid metabolism and energy homeostasis. Targeted inhibition of miR-33 increases plasma HDL (high-density lipoprotein) cholesterol and promotes atherosclerosis regression, in part, by enhancing reverse cholesterol transport and dampening plaque inflammation. However, how miR-33 reshapes the immune microenvironment of plaques remains poorly understood. Objective: To define how miR-33 inhibition alters the dynamic balance and transcriptional landscape of immune cells in atherosclerotic plaques. Methods and Results: We used single-cell RNA-sequencing of aortic CD45 + cells, combined with immunohistologic, morphometric, and flow cytometric analyses to define the changes in plaque immune cell composition, gene expression, and function following miR-33 inhibition. We report that anti–miR-33 treatment of Ldlr –/– mice with advanced atherosclerosis reduced plaque burden and altered the plaque immune cell landscape by shifting the balance of proatherosclerotic and antiatherosclerotic macrophage and T-cell subsets. By quantifying the kinetic processes that determine plaque macrophage burden, we found that anti–miR-33 reduced levels of circulating monocytes and splenic myeloid progenitors, decreased macrophage proliferation and retention, and promoted macrophage attrition by apoptosis and efferocytotic clearance. Single-cell RNA-sequencing of aortic arch plaques showed that anti–miR-33 reduced the frequency of MHCII hi (major histocompatibility complex II) inflammatory and Trem2 hi (Triggering Receptor Expressed On Myeloid Cells 2) metabolic macrophages, but not tissue-resident macrophages. Furthermore, anti–miR-33 led to derepression of distinct miR-33 target genes in the different macrophage subsets: in resident and Trem2 hi macrophages, anti–miR-33 relieved repression of miR-33 target genes involved in lipid metabolism (eg, Abca1 , Ncoa1, Ncoa2 , Crot ), whereas in MHCII hi macrophages, anti–miR-33 upregulated target genes involved in chromatin remodeling and transcriptional regulation. Anti–miR-33 also reduced the accumulation of aortic CD8+ T cells and CD4+ T-helper 1 cells, and increased levels of FoxP3+ (Forkhead Box P3) regulatory T cells in plaques, consistent with an immune-dampening effect on plaque inflammation. Conclusions: Our results provide insight into the immune mechanisms and cellular players that execute anti–miR-33’s atheroprotective actions in the plaque.

Psychological stress (PS) is associated with systemic inflammation and accelerates inflammatory disease progression (e.g., atherosclerosis). The mechanisms underlying stress-mediated inflammation and future health risk are poorly understood. Monocytes are key in sustaining systemic inflammation, and recent studies demonstrate that they maintain the memory of inflammatory insults, leading to a heightened inflammatory response upon rechallenge. We show that PS induces remodeling of the chromatin landscape and transcriptomic reprogramming of monocytes, skewing them to a primed hyperinflammatory phenotype. Monocytes from stressed mice and humans exhibit a characteristic inflammatory transcriptomic signature and are hyperresponsive upon stimulation with Toll-like receptor ligands. RNA and ATAC sequencing reveal that monocytes from stressed mice and humans exhibit activation of metabolic pathways (mTOR and PI3K) and reduced chromatin accessibility at mitochondrial respiration-associated loci. Collectively, our findings suggest that PS primes the reprogramming of myeloid cells to a hyperresponsive inflammatory state, which may explain how PS confers inflammatory disease risk.

Acute myocardial infarction rapidly increases blood neutrophils (

Rationale: Regression of atherosclerosis is an important clinical goal; however, the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, yet the numbers of these immunosuppressive cells decrease with disease progression, and whether they contribute to atherosclerosis regression is not known. Objective: We investigated the roles of Tregs in the resolution of atherosclerotic inflammation, tissue remodeling, and plaque contraction during atherosclerosis regression. Methods and Results: Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. Single-cell RNA-sequencing of plaque immune cells revealed that unlike Tregs from progressing plaques that expressed markers of natural Tregs derived from the thymus, Tregs in regressing plaques lacked Nrp1 expression, suggesting that they are induced in the periphery during lipid-lowering therapy. To test whether Tregs are required for resolution of atherosclerotic inflammation and plaque regression, Tregs were depleted using CD25 monoclonal antibody in atherosclerotic mice during apolipoprotein B antisense oligonucleotide-mediated lipid lowering. Morphometric analyses revealed that Treg depletion blocked plaque remodeling and contraction, and impaired hallmarks of inflammation resolution, including dampening of the T helper 1 response, alternative activation of macrophages, efferocytosis, and upregulation of specialized proresolving lipid mediators. Conclusions: Our data establish essential roles for Tregs in resolving atherosclerotic cardiovascular disease and provide mechanistic insight into the pathways governing plaque remodeling and regression of disease.

During obesity, macrophages infiltrate the visceral adipose tissue and promote inflammation that contributes to type II diabetes. Evidence suggests that the rewiring of cellular metabolism can regulate macrophage function. However, the metabolic programs that characterize adipose tissue macrophages (ATM) in obesity are poorly defined. Here, we demonstrate that ATM from obese mice exhibit metabolic profiles characterized by elevated glycolysis and oxidative phosphorylation, distinct from ATM from lean mice. Increased activation of HIF-1α in ATM of obese visceral adipose tissue resulted in induction of IL-1β and genes in the glycolytic pathway. Using a hypoxia-tracer, we show that HIF-1α nuclear translocation occurred both in hypoxic and non-hypoxic ATM suggesting that both hypoxic and pseudohypoxic stimuli activate HIF-1α and its target genes in ATM during diet-induced obesity. Exposure of macrophages to the saturated fatty acid palmitate increased glycolysis and HIF-1α expression, which culminated in IL-1β induction thereby simulating pseudohypoxia. Using mice with macrophage-specific targeted deletion of HIF-1α, we demonstrate the critical role of HIF-1α-derived from macrophages in regulating ATM accumulation, and local and systemic IL-1β production, but not in modulating systemic metabolic responses. Collectively, our data identify enhanced glycolysis and HIF-1α activation as drivers of low-grade inflammation in obesity.

Ley et al. provide a review of the technology and accomplishments of dynamic imaging of myeloid cells in atherosclerosis., Intravital imaging is an invaluable tool for understanding the function of cells in healthy and diseased tissues. It provides a window into dynamic processes that cannot be studied by other techniques. This review will cover the benefits and limitations of various techniques for labeling and imaging myeloid cells, with a special focus on imaging cells in atherosclerotic arteries. Although intravital imaging is a powerful tool for understanding cell function, it alone does not provide a complete picture of the cell. Other techniques, such as flow cytometry and transcriptomics, must be combined with intravital imaging to fully understand a cell's phenotype, lineage, and function.

Macrophages in the heart have dual roles in injury and repair after myocardial infarction, and understanding the two sides of this coin using traditional 'bulk cell' technologies has been challenging. By combining genetic fate-mapping and single-cell transcriptomics, a new study (Nat. Immunol. 2019;20:29-39) reveals how distinct macrophage populations expand and diverge across the healthy heart and after infarction.

Disruption of systemic homeostasis by either chronic or acute stressors, such as obesity(1) or surgery(2), alters cancer pathogenesis. Cancer patients, particularly those with breast cancer, can be at increased risk for cardiovascular disease due to treatment toxicity and changes in lifestyle behaviors(3–5). While elevated risk and incidence of cardiovascular events in breast cancer is well-established, whether such events impact cancer pathogenesis is not known. Here, we show that myocardial infarction (MI) accelerates breast cancer outgrowth and cancer-specific mortality in mice and humans. In mouse models of breast cancer, MI epigenetically reprogrammed Ly6C(high) monocytes in the bone marrow reservoir to an immunosuppressive phenotype that was maintained at the transcriptional level in monocytes in both the circulation and tumor. In parallel, MI increased circulating Ly6C(high) monocyte levels and recruitment to tumors, and depletion of these cells abrogated MI-induced tumor growth. Furthermore, early-stage breast cancer patients who experienced cardiovascular events after cancer diagnosis had increased risk of recurrence and cancer-specific death. These preclinical and clinical results demonstrate that MI induces alterations to systemic homeostasis, triggering cross-disease communication that accelerates breast cancer.

Observational findings suggest exercise is associated with improved outcomes in early-stage breast cancer. However, whether exercise has biological activity in patients with breast cancer has not been investigated. Preoperative window of opportunity studies provide a setting in which to test the short-term effects of novel treatment strategies on validated surrogates. See related article by Ligibel et al., p. 5398

Objective— Cholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. Approach and Results— Here, we identify oxysterol-binding protein-like 6 ( OSBPL6 ) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum–targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. Conclusions— These studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

Purpose To evaluate the effects of exercise therapy on cardiorespiratory fitness (CRF) in randomized controlled trials (RCTs) among patients with adult-onset cancer. Secondary objectives were to evaluate treatment effect modifiers, safety, and fidelity. Methods A systematic search of PubMed, Embase, Cumulative Index to Nursing and Allied Health Literature, and the Cochrane Library was conducted to identify RCTs that compared exercise therapy to a nonexercise control group. The primary end point was change in CRF as evaluated by peak oxygen consumption (VO2peak; in mL O2 × kg−1 × min−1) from baseline to postintervention. Subgroup analyses evaluated whether treatment effects differed as a function of exercise prescription (ie, modality, schedule, length, supervision), study characteristics (ie, intervention timing, primary cancer site), and publication year. Safety was defined as report of any adverse event (AE); fidelity was evaluated by rates of attendance, adherence, and loss to follow-up. Results Forty-eight unique RCTs that represented 3,632 patients (mean standard deviation age, 55 ± 7.5 years; 68% women); 1,990 (55%) and 1,642 (45%) allocated to exercise therapy and control/usual care groups, respectively, were evaluated. Exercise therapy was associated with a significant increase in CRF (+2.80 mL O2 × kg−1 × min−1) compared with no change (+0.02 mL O2 × kg−1 × min−1) in the control group (weighted mean differences, +2.13 mL O2 × kg−1 × min−1; 95% CI, 1.58 to 2.67; I2, 20.6; P < .001). No statistical significant differences were observed on the basis of any treatment effect modifiers. Thirty trials (63%) monitored AEs; a total of 44 AEs were reported. The mean standard deviation loss to follow-up, attendance, and adherence rates were 11% ± 13%, 84% ± 12%, and 88% ± 32%, respectively. Conclusion Exercise therapy is an effective adjunctive therapy to improve CRF in patients with cancer. Our findings support the recommendation of exercise therapy for patients with adult-onset cancer.

Previous studies have identified microRNA-33 (miR-33) as a key regulator of metabolic gene pathways involved in cholesterol and fatty acid homeostasis. Inhibition of miR-33 promotes the regression of atherosclerosis, in part, by enhancing plasma HDL and reverse cholesterol transport levels. However, it has become clear that miR-33 inhibition can also reduce plaque inflammation, independent of its effects on cholesterol homeostasis, yet the mechanisms of these actions are still not well understood. To investigate how miR-33 inhibition alters monocyte/macrophage dynamics during atherosclerosis regression, we fed Ldlr -/- mice a western diet for 14 weeks to establish plaques, after which mice were switched to chow diet and treated with anti-miR-33 or control anti-miR oligonucleotides for 4 weeks. As we reported previously, anti-miR-33 treatment increased plasma levels of HDL cholesterol by 30%, and concurrently reduced plaque size, macrophage content and necrotic area, compared to control anti-miR treatment. Analysis of the monocyte/macrophage kinetic processes contributing to macrophage burden in plaques revealed that anti-miR-33 treatment normalized the monocytosis associated with hypercholesterolemia. Mice treated with anti-miR33 had 40% fewer circulating monocytes than control anti-miR treated mice, and this was associated with a reduction of splenic common myeloid progenitor (CMP) cells. Surprisingly, monocyte tracking assays also revealed an increase in Ly6C hi monocyte recruitment into plaques of anti-miR-33 treated mice. Although somewhat counterintuitive, these data are consistent with recent findings that Ly6C hi monocytes are required for atherosclerosis regression and are a source of tissue reparative M2 macrophages. Indeed, we find that M2 macrophages, as well as atheroprotective regulatory T cells are enriched in plaques of anti-miR-33 treated mice. Collectively, our results provide insight into the mechanisms underlying anti-miR-33’s atheroprotective actions, which include both anti-inflammatory and cholesterol homeostatic effects.

Cardiovascular (CV) disease and cancer-specific mortality compete as the leading causes of mortality in women with early-stage breast cancer. Following treatment for early-stage disease, breast cancer patients are 5 times more likely to develop CV complications such as myocardial infarction (MI) compared to age-matched controls, due to the ‘cardiotoxic’ effects of anti-cancer therapies on CV tissues (e.g. chemotherapy, radiation). As CV disease and cancer progression share many common risk factors and etiologies, we hypothesized that CV disease may also influence cancer progression. To investigate this potential cross-disease communication, we developed a mouse model using the left anterior descending (LAD) coronary artery permanent ligation technique of MI in the E0771 orthotopic breast cancer model. LAD ligation significantly increased tumor growth over 20 days compared to sham surgery controls (SHAM) (p

Long non-coding RNAs (lncRNAs), once considered ‘genomic junk’, have been found to regulate diverse biological processes and their study continues to reveal novel insights into lncRNA functions. Recent studies revealed that some lncRNAs may harbor small open reading frames (ORFs) that code for functional micropeptides. While investigating an unannotated primate-specific lncRNA, lncVLDLR, that is altered in patients with type II diabetes and cardiovascular disease, we discovered a previously unrecognized ORF encoding a 44 amino acid micropeptide. In vitro transcription and translation of the IMP coding sequence in the presence of 35 S-methionine produced a single 8 kDa peptide, which we have named Inflammation-modulating MicroPeptide (IMP). To dissect IMP function, we focused on its amino acid sequence and putative structure. These analyses revealed high sequence homology between IMP and transcription factors such as NFκB, c-myb and zinc finger proteins, and the presence of a hydrophobic region with an LxxLL motif often found in transcriptional regulators. Circular dichroism spectroscopy of synthesized IMP predicted an intrinsically disordered peptide, which is a common characteristic of transcriptional coactivators. To investigate a potential role of IMP in regulating gene transcription, we cloned a MYC-epitope tag in-frame with IMP within the full-length transcript of lncVLDLR and expressed it in HEK293 cells. Immunofluorescence staining, and cell fractionation combined with western blotting, confirmed nuclear localization of IMP. RNA-seq analysis of THP1 macrophages overexpressing IMP revealed an increase in inflammatory genes, including cytokines and chemokines. Moreover, analysis of upstream regulators of these genes suggests that IMP may interact with KIX domain-containing transcriptional coactivators to regulate inflammatory gene expression. Together our data identify a novel human micropeptide, encoded within a putative lncRNA that is dysregulated in diabetes and cardiovascular disease, that regulates inflammatory gene transcription. Further characterization of IMP and its regulatory network may uncover novel opportunities for therapeutic intervention in cardiovascular and other inflammatory diseases.

After activation, cells of the myeloid lineage undergo robust metabolic transitions, as well as discrete epigenetic changes, that can dictate both ongoing and future inflammatory responses. In atherosclerosis, in which macrophages play central roles in the initiation, growth, and ultimately rupture of arterial plaques, altered metabolism is a key feature that dictates macrophage function and subsequent disease progression. This Review explores how factors central to the plaque microenvironment (for example, altered cholesterol metabolism, oxidative stress, hypoxia, apoptotic and necrotic cells, and hyperglycemia) shape the metabolic rewiring of macrophages in atherosclerosis as well as how these metabolic shifts in turn alter macrophage immune-effector and tissue-reparative functions. Finally, this overview offers insight into the challenges and opportunities of harnessing metabolism to modulate aberrant macrophage responses in disease.

The field of exercise-oncology has increased dramatically over the past two decades, with close to 100 published studies investigating the efficacy of structured exercise training interventions in patients with cancer. Of interest, despite considerable differences in study population and primary study end point, the vast majority of studies have tested the efficacy of an exercise prescription that adhered to traditional guidelines consisting of either supervised or home-based endurance (aerobic) training or endurance training combined with resistance training, prescribed at a moderate intensity (50-75% of a predetermined physiological parameter, typically age-predicted heart rate maximum or reserve), for two to three sessions per week, for 10 to 60 min per exercise session, for 12 to 15 weeks. The use of generic exercise prescriptions may, however, be masking the full therapeutic potential of exercise treatment in the oncology setting. Against this background, this opinion paper provides an overview of the fundamental tenets of human exercise physiology known as the principles of training, with specific application of these principles in the design and conduct of clinical trials in exercise-oncology research. We contend that the application of these guidelines will ensure continued progress in the field while optimizing the safety and efficacy of exercise treatment following a cancer diagnosis.

The integrity and composition of the tumour microenvironment (TME) is highly plastic, undergoing constant remodelling in response to instructive signals derived from alterations in the availability and nature of systemic host factors. This 'systemic milieu' is directly modulated by host exposure to modifiable lifestyle factors such as exercise. Host exposure to regular exercise markedly reduces the risk of the primary development of several cancers and might improve clinical outcomes following a diagnosis of a primary disease. However, the molecular mechanisms that underpin the apparent antitumour effects of exercise are poorly understood. In this Opinion article, we explore the putative effects of exercise in reprogramming the interaction between the host and the TME. Specifically, we speculate on the possible effects of exercise on reprogramming 'distant' tissue microenvironments (those not directly involved in the exercise response) by analysing how alterations in the systemic milieu might modulate key TME components to influence cancer hallmarks.

Cholesterol homeostasis is fundamental to human health, and is thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. Here, we identify oxysterol binding protein-like 6 ( OSBPL6 ) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by LXR and in response to cholesterol loading, and in mice and non-human primates by western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains a pleckstrin homology membrane-targeting domain and a FFAT domain predicted to interact with VAP proteins in the ER. Indeed, subcellular localization studies showed that ORP6 is associated with the plasma membrane, early endosomes, lysosomes and ER, suggesting a putative role for ORP6 in cholesterol trafficking between these compartments. Consistent with this, siRNA-mediated knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures. As a consequence, esterification of cholesterol at the ER is reduced, triggering activation of SREBP2. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux to apoA1 in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of HDL cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. Collectively, these studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

Advances in detection and supportive care strategies have led to improvements in cancer-specific and overall survival after a diagnosis of early-stage breast cancer. These improvements, however, are associated with an increase in competing forms of morbidity and mortality particularly cardiovascular disease (CVD). Indeed, in certain subpopulations of patients after early breast cancer, CVD is not only the leading cause of mortality but these women also have an increased risk of CVD-specific morbidity, including elevated incidence of coronary artery disease and heart failure (HF) compared to sex- and age-matched counterparts. Exercise treatment is established as the cornerstone of primary and secondary prevention of CVD in multiple clinical populations. The potential benefits of exercise treatment to modulate CVD or CVD risk factors before, immediately after, or in the months/years following adjuvant therapy for early-stage breast cancer has received limited attention. Here, we discuss the risk and extent of CVD in breast cancer patients, review the pathogenesis of CVD, and highlight existing evidence from select clinical trials investigating the efficacy of structured exercise treatment across the CVD continuum in early breast cancer.

Anthracycline-containing chemotherapy (Anth-C) is associated with long-term cardiovascular mortality. Although cardiovascular risk assessment has traditionally focused on the heart, evidence has demonstrated that vascular dysfunction also occurs during and up to 1 year following Anth-C. Whether vascular dysfunction persists long-term or negatively influences cardiac function remains unknown. Hence, the present study evaluated ventricular-arterial coupling, in concert with measures of vascular structure and function, in the years following Anth-C.Arterial elastance (Ea), end-systolic elastance (Ees), and ventricular-arterial coupling (Ea/Ees) were measured during rest and exercise using echocardiography. Resting vascular function (flow-mediated dilation) and structure (carotid intima-media thickness, arterial stiffness) were also measured.Thirty breast cancer survivors (6.5 ± 3.6 years after Anth-C) with normal left ventricular ejection fraction (LVEF) (60% ± 6%) and 30 matched controls were studied. At rest, no differences were found in Ea, Ees, Ea/Ees, or LVEF between groups. The normal exercise-induced increase in Ees was attenuated in survivors at 50% and 75% of maximal workload (p.01). Ea/Ees was also higher at all workloads in the survivors compared with the controls (p.01). No differences in vascular structure and function were observed between the two groups (p.05).In the years after Anth-C, ventricular-arterial coupling was significantly attenuated during exercise, primarily owing to decreased LV contractility (indicated by a reduced Ees). This subclinical dysfunction appears to be isolated to the heart, as no differences in Ea were observed. The previously reported adverse effects of Anth-C on the vasculature appear to not persist in the years after treatment, as vascular structure and function were comparable to controls.Anthracycline-induced cardiotoxicity results in significantly impaired ventricular-arterial coupling in the years following chemotherapy, owing specifically to decreased left ventricular contractility. This subclinical dysfunction was identified only under exercise stress. A comprehensive evaluation of vascular structure and function yielded no differences between those treated with anthracyclines and controls. Combined with a stress stimulus, ventricular-arterial coupling might hold significant value beyond characterization of integrative cardiovascular function, in particular as a part of a risk-stratification strategy after anthracycline-containing chemotherapy. Although vascular function and structure were not different in this cohort, this does not undermine the importance of identifying vascular (dys)function in this population, because increases in net arterial load during exercise might amplify the effect of reductions in contractility on cardiovascular function after anthracycline-containing chemotherapy.

BACKGROUND: Monoclonal antibodies (mAb) that target CTLA-4, a negative regulator of T-cell activation, have significantly extended survival of patients with metastatic melanoma. However the number of responders remain low. We have previously shown in the 4T1 mouse breast tumor model that resistance to α-CTLA-4 therapy can be overcome by concurrent local radiotherapy (RT) (Demaria et al Clin Can Res 2005). Multiple effects of RT on the tumor microenvironment contributed to its ability to sensitize tumors to anti-CTLA-4 treatment, including enhanced homing of effector T cells to the tumor and formation of stable immune synapses between CTL and tumor cells.Importantly, CD8 T cell responses to the endogenous tumor antigen gp70 were seen only in mice treated with the combination, consistent with the hypothesis that RT generates an in situ vaccine and CTLA-4 expands primed T cells. Here we used next generation sequencing of complementarity determining region 3 (CDR3) from rearranged T cell receptor (TCR) variable beta (Vβ) chains to obtain a comprehensive picture of the changes in the repertoire of infiltrating lymphocytes that occur during CD8 T cell-mediated tumor rejection. METHODS: BALB/c mice were inoculated with 4T1 cells and thirteen days later, when tumors became palpable, randomly assigned to one of 4 treatment groups (N = 5 mice/group): control, RT alone, α-CTLA-4 alone or RT+anti-CTLA-4. RT was given in 2 fractions of 12 Gy on days 13 and 14 post-tumor inoculation. α-CTLA-4 mAb (Clone 9H10) was given i.p. on days 15, 18 and 21. Tumors were harvested on day 22 for high throughput sequencing of TCRβ CDR3 regions performed using the ImmunoSEQ platform. RESULTS: We observed distinct and non-overlapping effects of RT and immunotherapy on TIL repertoire. CTLA-4 blockade increased clonality and significantly expanded the top 5 most frequent clonotypes. On the other hand, RT increased TIL numbers and broadened their repertoire by selectively expanding the top 6-20 clonotypes. Importantly, analysis of Vβ/Jβ usage landscape showed that the combined treatment generated the most dramatic changes from baseline with the expansion of several unique Vβ/Jβ combinations not otherwise seen in tumors treated with RT or CTLA-4 blockade as single agents. Significantly reduced diversity of the TCRβ profile seen only with combination therapy is consistent with antigen-driven TIL clonal expansion.CONCLUSIONS: Overall, tumor rejection induced by RT+anti-CTLA-4 is associated with both quantitative and qualitative changes in TIL repertoire. Data suggests that a broader repertoire of tumor-specific T cells may be critical for tumor rejection, and is achieved by coupling the effects of RT, which promotes priming of T cells specific for endogenous tumor antigens, and CTLA-4 blockade, which drives expansion of selected clones. These results have important implications for the therapeutic synergy of radiation and CTLA-4 blockade. Citation Format: Karsten Pilones, Graeme Koelwyn, Ryan Emerson, Silvia Formenti, Harlan Robins, Sandra Demaria. Unique changes in the TCR repertoire of tumor-infiltrating lymphocytes underlie the synergy of radiotherapy with CTLA-4 blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2856. doi:10.1158/1538-7445.AM2015-2856