Endothelial-to-Mesenchymal Transition Contributes to Accelerated Atherosclerosis in Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Atherosclerosis is the main medical problem in Hutchinson-Gilford progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe-deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear. METHODS: Apoe- or Ldlr-deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFß (transforming growth factor ß) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFß1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root. RESULTS: Both ubiquitous and VSMC-specific progerin expression in Apoe-null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFß1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype. CONCLUSIONS: Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFß1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis.

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.

SpatialDDLS: an R package to deconvolute spatial transcriptomics data using neural networks.

SUMMARY: Spatial transcriptomics has changed our way to study tissue structure and cellular organization. However, there are still limitations in its resolution, and most available platforms do not reach a single cell resolution. To address this issue, we introduce SpatialDDLS, a fast neural network-based algorithm for cell type deconvolution of spatial transcriptomics data. SpatialDDLS leverages single-cell RNA sequencing data to simulate mixed transcriptional profiles with predefined cellular composition, which are subsequently used to train a fully connected neural network to uncover cell type diversity within each spot. By comparing it with two state-of-the-art spatial deconvolution methods, we demonstrate that SpatialDDLS is an accurate and fast alternative to the available state-of-the art tools. AVAILABILITY AND IMPLEMENTATION: The R package SpatialDDLS is available via CRAN-The Comprehensive R Archive Network: https://CRAN.R-project.org/package=SpatialDDLS. A detailed manual of the main functionalities implemented in the package can be found at https://diegommcc.github.io/SpatialDDLS.

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

BACKGROUND: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. METHODS: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. RESULTS: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. CONCLUSIONS: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.

Heteroplasmy of Wild-Type Mitochondrial DNA Variants in Mice Causes Metabolic Heart Disease With Pulmonary Hypertension and Frailty.

BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.

Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing.

Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

Functional interplay between c-Myc and Max in B lymphocyte differentiation.

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

The zebrafish reference genome sequence and its relationship to the human genome.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

Meis1 coordinates a network of genes implicated in eye development and microphthalmia.

Microphthalmos is a rare congenital anomaly characterized by reduced eye size and visual deficits of variable degree. Sporadic and hereditary microphthalmos have been associated with heterozygous mutations in genes fundamental for eye development. Yet, many cases are idiopathic or await the identification of molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adult mice. By combining analysis of Meis1 loss-of-function and conditional Meis1 functional rescue with ChIP-seq and RNA-seq approaches we show that, in contrast to its preferential association with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbx-independent sites during optic cup development. In the eye primordium, Meis1 coordinates, in a dose-dependent manner, retinal proliferation and differentiation by regulating genes responsible for human microphthalmia and components of the Notch signaling pathway. In addition, Meis1 is required for eye patterning by controlling a set of eye territory-specific transcription factors, so that in Meis1(-/-) embryos boundaries among the different eye territories are shifted or blurred. We propose that Meis1 is at the core of a genetic network implicated in eye patterning/microphthalmia, and represents an additional candidate for syndromic cases of these ocular malformations.

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium.

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.

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.

Endothelial YAP/TAZ activation promotes atherosclerosis in a mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare disease caused by the expression of progerin, an aberrant protein produced by a point mutation in the LMNA gene. HGPS patients show accelerated aging and die prematurely mainly from complications of atherosclerosis such as myocardial infarction, heart failure, or stroke. However, the mechanisms underlying HGPS vascular pathology remain ill defined. We used single-cell RNA sequencing to characterize the aorta in progerin-expressing LmnaG609G/G609G mice and wild-type controls, with a special focus on endothelial cells (ECs). HGPS ECs showed gene expression changes associated with extracellular matrix alterations, increased leukocyte extravasation, and activation of the yes-associated protein 1/transcriptional activator with PDZ-binding domain (YAP/TAZ) mechanosensing pathway, all validated by different techniques. Atomic force microscopy experiments demonstrated stiffer subendothelial extracellular matrix in progeroid aortas, and ultrasound assessment of live HGPS mice revealed disturbed aortic blood flow, both key inducers of the YAP/TAZ pathway in ECs. YAP/TAZ inhibition with verteporfin reduced leukocyte accumulation in the aortic intimal layer and decreased atherosclerosis burden in progeroid mice. Our findings identify endothelial YAP/TAZ signaling as a key mechanism of HGPS vascular disease and open a new avenue for the development of YAP/TAZ targeting drugs to ameliorate progerin-induced atherosclerosis.

The G4 resolvase Dhx36 modulates cardiomyocyte differentiation and ventricular conduction system development.

Extensive genetic studies have elucidated cardiomyocyte differentiation and associated gene networks using single-cell RNA-seq, yet the intricate transcriptional mechanisms governing cardiac conduction system (CCS) development and working cardiomyocyte differentiation remain largely unexplored. Here we show that mice deleted for Dhx36 (encoding the Dhx36 helicase) in the embryonic or neonatal heart develop overt dilated cardiomyopathy, surface ECG alterations related to cardiac impulse propagation, and (in the embryonic heart) a lack of a ventricular conduction system (VCS). Heart snRNA-seq and snATAC-seq reveal the role of Dhx36 in CCS development and in the differentiation of working cardiomyocytes. Dhx36 deficiency directly influences cardiomyocyte gene networks by disrupting the resolution of promoter G-quadruplexes in key cardiac genes, impacting cardiomyocyte differentiation and CCS morphogenesis, and ultimately leading to dilated cardiomyopathy and atrioventricular block. These findings further identify crucial genes and pathways that regulate the development and function of the VCS/Purkinje fiber (PF) network.

Evolution of the mammalian embryonic pluripotency gene regulatory network.

Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

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.

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.