Unidirectional association of clonal hematopoiesis with atherosclerosis development.

Clonal hematopoiesis, a condition in which acquired somatic mutations in hematopoietic stem cells lead to the outgrowth of a mutant hematopoietic clone, is associated with a higher risk of hematological cancer and a growing list of nonhematological disorders, most notably atherosclerosis and associated cardiovascular disease. However, whether accelerated atherosclerosis is a cause or a consequence of clonal hematopoiesis remains a matter of debate. Some studies support a direct contribution of certain clonal hematopoiesis-related mutations to atherosclerosis via exacerbation of inflammatory responses, whereas others suggest that clonal hematopoiesis is a symptom rather than a cause of atherosclerosis, as atherosclerosis or related traits may accelerate the expansion of mutant hematopoietic clones. Here we combine high-sensitivity DNA sequencing in blood and noninvasive vascular imaging to investigate the interplay between clonal hematopoiesis and atherosclerosis in a longitudinal cohort of healthy middle-aged individuals. We found that the presence of a clonal hematopoiesis-related mutation confers an increased risk of developing de novo femoral atherosclerosis over a 6-year period, whereas neither the presence nor the extent of atherosclerosis affects mutant cell expansion during this timeframe. These findings indicate that clonal hematopoiesis unidirectionally promotes atherosclerosis, which should help translate the growing understanding of this condition into strategies for the prevention of atherosclerotic cardiovascular disease in individuals exhibiting clonal hematopoiesis.

Cholesterol lowering depletes atherosclerotic lesions of smooth muscle cell-derived fibromyocytes and chondromyocytes.

Drugs that lower plasma apolipoprotein B (ApoB)-containing lipoproteins are central to treating advanced atherosclerosis and provide partial protection against clinical events. Previous research showed that lowering ApoB-containing lipoproteins stops plaque inflammation, but how these drugs affect the heterogeneous population of plaque cells derived from smooth muscle cells (SMCs) is unknown. SMC-derived cells are the main cellular component of atherosclerotic lesions and the source of structural components that determine the size of plaques and their propensity to rupture and trigger thrombosis, the proximate cause of heart attack and stroke. Using lineage tracing and single-cell techniques to investigate the full SMC-derived cellular compartment in progressing and regressing plaques in mice, here we show that lowering ApoB-containing lipoproteins reduces nuclear factor kappa-light-chain-enhancer of activated B cells signaling in SMC-derived fibromyocytes and chondromyocytes and leads to depletion of these abundant cell types from plaques. These results uncover an important mechanism through which cholesterol-lowering drugs can achieve plaque regression.

Incongruence between transcriptional and vascular pathophysiological cell states.

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

Cardiovascular toxicity of checkpoint inhibitors: review of associated toxicity and design of the Spanish Immunotherapy Registry of Cardiovascular Toxicity.

Immune checkpoint inhibitors (ICI) have changed the prognosis of many tumors. However, concerning associated cardiotoxicity has been reported. Little is known about the real-life incidence-specific surveillance protocols or the translational correlation between the underlying mechanisms and the clinical presentation of ICI-induced cardiotoxicity. The lack of data from prospective studies led us to review the current knowledge and to present the creation of the Spanish Immunotherapy Registry of Cardiovascular Toxicity (SIR-CVT), a prospective registry of patients receiving ICI that aims to examine the role of hsa-miR-Chr8:96, (a specific serum biomarker of myocarditis) in the early diagnosis of ICI-induced myocarditis. An exhaustive prospective cardiac imaging study will be performed before and during the first 12 months of treatment. The correlation between clinical, imaging, and immunologic parameters may improve our understanding of ICI-induced cardiotoxicity and enable simpler surveillance protocols. We assess ICI-induced cardiovascular toxicity and describe the rationale of the SIR-CVT.

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation.

Here we explored the role of interleukin-1ß (IL-1ß) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1ß monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1ß/IL-1rn levels under steady-state, and that loss of repression of IL-1ß signaling may underlie pre-leukemic lesion and AML progression.

Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Herein, we show that dual deficiency for hematopoietic RXRα and RXRß induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRα and RXRß maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRα;RXRß-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRα;RXRß-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.

Clonal hematopoiesis is not prevalent in Hutchinson-Gilford progeria syndrome.

Clonal hematopoiesis of indeterminate potential (CHIP), defined as the presence of somatic mutations in cancer-related genes in blood cells in the absence of hematological cancer, has recently emerged as an important risk factor for several age-related conditions, especially cardiovascular disease. CHIP is strongly associated with normal aging, but its role in premature aging syndromes is unknown. Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare genetic condition driven by the accumulation of a truncated form of the lamin A protein called progerin. HGPS patients exhibit several features of accelerated aging and typically die from cardiovascular complications in their early teens. Previous studies have shown normal hematological parameters in HGPS patients, except for elevated platelets, and low levels of lamin A expression in hematopoietic cells relative to other cell types in solid tissues, but the prevalence of CHIP in HGPS remains unexplored. To investigate the potential role of CHIP in HGPS, we performed high-sensitivity targeted sequencing of CHIP-related genes in blood DNA samples from a cohort of 47 HGPS patients. As a control, the same sequencing strategy was applied to blood DNA samples from middle-aged and elderly individuals, expected to exhibit a biological age and cardiovascular risk profile similar to HGPS patients. We found that CHIP is not prevalent in HGPS patients, in marked contrast to our observations in individuals who age normally. Thus, our study unveils a major difference between HGPS and normal aging and provides conclusive evidence that CHIP is not frequent in HGPS and, therefore, is unlikely to contribute to the pathophysiology of this accelerated aging syndrome.

Functional Impact and Regulation of Alternative Splicing in Mouse Heart Development and Disease.

Alternative splicing (AS) plays a major role in the generation of transcript diversity. In the heart, roles have been described for some AS variants, but the global impact and regulation of AS patterns are poorly understood. Here, we studied the AS profiles in heart disease, their relationship with heart development, and the regulatory mechanisms controlling AS dynamics in the mouse heart. We found that AS profiles characterized the different groups and that AS and gene expression changes affected independent genes and biological functions. Moreover, AS changes, specifically in heart disease, were associated with potential protein-protein interaction changes. While developmental transitions were mainly driven by the upregulation of MBNL1, AS changes in disease were driven by a complex regulatory network, where PTBP1 played a central role. Indeed, PTBP1 over-expression was sufficient to induce cardiac hypertrophy and diastolic dysfunction, potentially by perturbing AS patterns.

Behavioural immune landscapes of inflammation.

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

[Figure: see text].

Transcriptional epigenetic regulation of Fkbp1/Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer.

BACKGROUND: In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. METHODS: We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. RESULTS: We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. CONCLUSIONS: Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient's progression and therapeutic response.

Clonal Hematopoiesis and Risk of Progression of Heart Failure With Reduced Left Ventricular Ejection Fraction.

BACKGROUND: Clonal hematopoiesis driven by somatic mutations in hematopoietic cells, frequently called clonal hematopoiesis of indeterminate potential (CHIP), has been associated with adverse cardiovascular outcomes in population-based studies and in patients with ischemic heart failure (HF) and reduced left ventricular ejection fraction (LVEF). Yet, the impact of CHIP on HF progression, including nonischemic etiology, is unknown. OBJECTIVES: The purpose of this study was to assess the clinical impact of clonal hematopoiesis on HF progression irrespective of its etiology. METHODS: The study cohort comprised 62 patients with HF and LVEF <45% (age 74 ± 7 years, 74% men, 52% nonischemic, and LVEF 30 ± 8%). Deep sequencing was used to detect CHIP mutations with a variant allelic fraction >2% in 54 genes. Patients were followed for at least 3.5 years for various adverse events including death, HF-related death, and HF hospitalization. RESULTS: CHIP mutations were detected in 24 (38.7%) patients, without significant differences in all-cause mortality (p = 0.151). After adjusting for risk factors, patients with mutations in either DNA methyltransferase 3 alpha (DNMT3A) or Tet methylcytosine dioxygenase 2 (TET2) exhibited accelerated HF progression in terms of death (hazard ratio [HR]: 2.79; 95% confidence interval [CI]: 1.31 to 5.92; p = 0.008), death or HF hospitalization (HR: 3.84; 95% CI: 1.84 to 8.04; p < 0.001) and HF-related death or HF hospitalization (HR: 4.41; 95% CI: 2.15 to 9.03; p < 0.001). In single gene-specific analyses, somatic mutations in DNMT3A or TET2 retained prognostic significance with regard to HF-related death or HF hospitalization (HR: 4.50; 95% CI: 2.07 to 9.74; p < 0.001, for DNMT3A mutations; HR: 3.18; 95% CI: 1.52 to 6.66; p = 0.002, for TET2 mutations). This association remained significant irrespective of ischemic/nonischemic etiology. CONCLUSIONS: Somatic mutations that drive clonal hematopoiesis are common among HF patients with reduced LVEF and are associated with accelerated HF progression regardless of etiology.

Harmine and Piperlongumine Revert TRIB2-Mediated Drug Resistance.

Therapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.

Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Identification of a peripheral blood gene signature predicting aortic valve calcification.

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9, 13, and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.

Sox17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels.

RATIONALE: The molecular mechanisms underlying the formation of coronary arteries during development and during cardiac neovascularization after injury are poorly understood. However, a detailed description of the relevant signaling pathways and functional TFs (transcription factors) regulating these processes is still incomplete. OBJECTIVE: The goal of this study is to identify novel cardiac transcriptional mechanisms of coronary angiogenesis and vessel remodeling by defining the molecular signatures of coronary vascular endothelial cells during these complex processes. METHODS AND RESULTS: We demonstrate that Nes-gfp and Nes-CreERT2 transgenic mouse lines are novel tools for studying the emergence of coronary endothelium and targeting sprouting coronary vessels (but not ventricular endocardium) during development. Furthermore, we identify Sox17 as a critical TF upregulated during the sprouting and remodeling of coronary vessels, visualized by a specific neural enhancer from the Nestin gene that is strongly induced in developing arterioles. Functionally, genetic-inducible endothelial deletion of Sox17 causes deficient cardiac remodeling of coronary vessels, resulting in improper coronary artery formation. CONCLUSIONS: We demonstrated that Sox17 TF regulates the transcriptional activation of Nestin's enhancer in developing coronary vessels while its genetic deletion leads to inadequate coronary artery formation. These findings identify Sox17 as a critical regulator for the remodeling of coronary vessels in the developing heart.

Caveolin1 and YAP drive mechanically induced mesothelial to mesenchymal transition and fibrosis.

Despite their emerging relevance to fully understand disease pathogenesis, we have as yet a poor understanding as to how biomechanical signals are integrated with specific biochemical pathways to determine cell behaviour. Mesothelial-to-mesenchymal transition (MMT) markers colocalized with TGF-ß1-dependent signaling and yes-associated protein (YAP) activation across biopsies from different pathologies exhibiting peritoneal fibrosis, supporting mechanotransduction as a central driving component of these class of fibrotic lesions and its crosstalk with specific signaling pathways. Transcriptome and proteome profiling of the response of mesothelial cells (MCs) to linear cyclic stretch revealed molecular changes compatible with bona fide MMT, which (i) overlapped with established YAP target gene subsets, and were largely dependent on endogenous TGF-ß1 signaling. Importantly, TGF-ß1 blockade blunts the transcriptional upregulation of these gene signatures, but not the mechanical activation and nuclear translocation of YAP per se. We studied the role therein of caveolin-1 (CAV1), a plasma membrane mechanotransducer. Exposure of CAV1-deficient MCs to cyclic stretch led to a robust upregulation of MMT-related gene programs, which was blunted upon TGF-ß1 inhibition. Conversely, CAV1 depletion enhanced both TGF-ß1 and TGFBRI expression, whereas its re-expression blunted mechanical stretching-induced MMT. CAV1 genetic deficiency exacerbated MMT and adhesion formation in an experimental murine model of peritoneal ischaemic buttons. Taken together, these results support that CAV1-YAP/TAZ fine-tune the fibrotic response through the modulation of MMT, onto which TGF-ß1-dependent signaling coordinately converges. Our findings reveal a cooperation between biomechanical and biochemical signals in the triggering of MMT, representing a novel potential opportunity to intervene mechanically induced disorders coursing with peritoneal fibrosis, such as post-surgical adhesions.