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Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNALINC00607to be highly enriched in human endothelial cells.LINC00607was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout ofLINC00607attenuated VEGF-A-induced angiogenic sprouting.LINC00607knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression ofLINC00607in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq afterLINC00607knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically,LINC00607interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout ofBRG1in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion,LINC00607is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

AIMS: Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis.METHODS & RESULTS: Myeloid specific PHD knockout (PHDko) mice were obtained via bone marrow transplantation (PHD1ko, PHD3ko) or conditional knockdown through lysozyme M-driven Cre recombinase (PHD2cko). Mice were fed high cholesterol diet for 6-12 weeks to induce atherosclerosis. Aortic root plaque size was significantly augmented 2.6-fold in PHD2cko, and 1.4-fold in PHD3ko compared to controls, but was unchanged in PHD1ko mice. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, showed enhanced HIF1α/BNIP3 signaling, which was validated in vitro by siRNA silencing. Human plaque BNIP3 mRNA was positively associated with plaque necrotic core size, suggesting similar pro-apoptotic effects in human. Further, PHD2cko plaques displayed enhanced fibrosis, while macrophage collagen breakdown by matrix metalloproteinases, collagen production and proliferation were unaltered. Instead, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. In silico analysis of macrophage-fibroblast communication predicted SPP1 (osteopontin) signaling as regulator, which was corroborated by enhanced plaque SPP1 protein in vivo. Increased SPP1 mRNA expression upon PHD2cko was preferentially observed in foamy plaque macrophages expressing "triggering receptor expressed on myeloid cells-2" (TREM2hi) evidenced by single-cell RNA, but not in neutrophils. This confirmed enhanced fibrotic signaling by PHD2cko macrophages to fibroblasts, in vitro as well as in vivo.CONCLUSION: Myeloid PHD2cko and PHD3ko enhanced atherosclerotic plaque growth and macrophage apoptosis, while PHD2cko macrophages further activated collagen secretion by fibroblasts in vitro, likely via paracrine SPP1 signaling through TREM2hi macrophages.TRANSLATIONAL OUTLOOK: This study shows that myeloid PHD isoforms PHD2 and PHD3 worsen plaque characteristics and phenotype, such as plaque size, macrophage accumulation, apoptosis, and collagen accumulation in mice. We show both direct effects on macrophages and paracrine effects of macrophage PHD2 loss on vessel wall fibroblast populations. Broad spectrum-PHD inhibitors, e.g. Roxadustat, are currently being prescribed to chronic kidney disease patients, who are already at risk for cardiovascular disease. When considering this study and the pro-fibrotic and pro-apoptotic effects we report, broad PHD inhibition may therefore be sub-optimal and more targeted PHD inhibition of PHD1 should be considered.

BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA ( INKILN ). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA–protein and protein–protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKIL N expression and function in ligation injury–induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1β–induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury–induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.

Aims Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. Methods and results Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. Conclusion We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.

Rationale: In the microenvironment of atherosclerotic lesions, vascular smooth muscle cells (vSMCs) switch to a dedifferentiated state but the underlying molecular mechanisms driving this switch are not fully understood. Long noncoding RNAs (lncRNAs) are dysregulated during vascular pathology, but relatively little is known about their involvement in controlling vSMCs function. Cardiac mesoderm enhancer-associated noncoding RNA (CARMN) is a lncRNA located immediately upstream of the microRNAs-143 and -145 (miR-143 and miR-145 ), both involved in vSMCs function. Objective: We investigated the role of the lncRNA CARMN, independent from miR-143 and miR-145, as a potential regulator of vSMC phenotypes in vitro and the consequences of its loss during the development of atherosclerosis in vivo. We hypothesized that loss of CARMN is a primary event controlling the functional switch towards proatherogenic vSMC phenotype and accelerates the development of the plaques in vivo. Method and Results: Expression of CARMN lncRNA was silenced using locked nucleic acids antisense oligonucleotides (GapmeRs) in human coronary arterial smooth muscle cells, revealing that GapmeR-mediated loss of CARMN negatively affects miR-143 and miR-145 microRNA expression. RNA sequencing of CARMN-depleted human coronary arterial smooth muscle cells revealed large transcriptomic changes, associated with vSMC proliferation, migration, inflammation, lipid metabolism, and dedifferentiation. The use of miR-143 and miR-145 mimics revealed that CARMN regulates human coronary arterial smooth muscle cell proliferation in a microRNA-independent manner. In humans and mice, CARMN and associated microRNAs were downregulated in advanced versus early atherosclerotic lesions. Using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) knockout approach, we explored the implications of CARMN depletion during atherosclerosis in vivo. Consistent with in vitro results, the knockout of CARMN impaired the expression of miR-143 and miR-145 under homeostatic conditions. Importantly, when atherosclerosis was induced in these mice, CARMN knockout increased the volume, size, proinflammatory Lgals3 (galectin 3)-expressing cells content, and altered plaque composition, yielding an advanced phenotype. Conclusions: We identified the early loss of CARMN lncRNA as critical event which primes vSMCs towards a proatherogenic phenotype in vitro and accelerates the development of atherosclerosis in vivo.

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1.

OBJECTIVES: The aim of this study was to describe the potential of 18F-sodium fluoride ( 18F-NaF) positron emission tomography (PET) to identify graft vasculopathy and to investigate the influence of coronary artery bypass graft (CABG) surgery on native coronary artery disease activity and progression. BACKGROUND: As well as developing graft vasculopathy, CABGs have been proposed to accelerate native coronary atherosclerosis.METHODS: Patients with established coronary artery disease underwent baseline 18F-NaF PET, coronary artery calcium scoring, coronary computed tomographic angiography, and 1-year repeat coronary artery calcium scoring. Whole-vessel coronary microcalcification activity (CMA) on 18F-NaF PET and change in calcium scores were quantified in patients with and without CABG surgery. RESULTS: Among 293 participants (mean age 65 ± 9 years, 84% men), 48 (16%) underwent CABG surgery 2.7 years [IQR: 1.4-10.4 years] previously. Although all arterial and the majority (120 of 128 [94%]) of vein grafts showed no 18F-NaF uptake, 8 saphenous vein grafts in 7 subjects had detectable CMA. Bypassed native coronary arteries had 3 times higher CMA values (2.1 [IQR: 0.4-7.5] vs 0.6 [IQR: 0-2.7]; P < 0.001) and greater progression of 1-year calcium scores (118 Agatston unit [IQR: 48-194 Agatston unit] vs 69 [IQR: 21-142 Agatston unit]; P = 0.01) compared with patients who had not undergone CABG, an effect confined largely to native coronary plaques proximal to the graft anastomosis. In sensitivity analysis, bypassed native coronary arteries had higher CMA (2.0 [IQR: 0.4-7.5] vs 0.8 [IQR: 0.3-3.2]; P < 0.001) and faster disease progression (24% [IQR: 16%-43%] vs 8% [IQR: 0%-24%]; P = 0.002) than matched patients (n = 48) with comparable burdens of coronary artery disease and cardiovascular comorbidities in the absence of bypass grafting. CONCLUSIONS: Native coronary arteries that have been bypassed demonstrate increased disease activity and more rapid disease progression than nonbypassed arteries, an observation that appears independent of baseline atherosclerotic plaque burden. Microcalcification activity is not a dominant feature of graft vasculopathy.

Proceedings of the National Academy of Sciences of the United States of America 119(14), e2121133119 (2022). doi:10.1073/pnas.2121133119, Published by National Acad. of Sciences, Washington, DC

Background: Type 2 myocardial infarction is caused by myocardial oxygen supply-demand imbalance, and its diagnosis is increasingly common with the advent of high-sensitivity cardiac troponin assays. Although this diagnosis is associated with poor outcomes, widespread uncertainty and confusion remain among clinicians as to how to investigate and manage this heterogeneous group of patients with type 2 myocardial infarction. Methods: In a prospective cohort study, 8064 consecutive patients with increased cardiac troponin concentrations were screened to identify patients with type 2 myocardial infarction. We excluded patients with frailty or renal or hepatic failure. All study participants underwent coronary (invasive or computed tomography angiography) and cardiac (magnetic resonance or echocardiography) imaging, and the underlying causes of infarction were independently adjudicated. The primary outcome was the prevalence of coronary artery disease. Results: In 100 patients with a provisional diagnosis of type 2 myocardial infarction (median age, 65 years [interquartile range, 55–74 years]; 43% women), coronary and cardiac imaging reclassified the diagnosis in 7 patients: type 1 or 4b myocardial infarction in 5 and acute myocardial injury in 2 patients. In those with type 2 myocardial infarction, median cardiac troponin I concentrations were 195 ng/L (interquartile range, 62–760 ng/L) at presentation and 1165 ng/L (interquartile range, 277–3782 ng/L) on repeat testing. The prevalence of coronary artery disease was 68% (63 of 93), which was obstructive in 30% (28 of 93). Infarct-pattern late gadolinium enhancement or regional wall motion abnormalities were observed in 42% (39 of 93), and left ventricular systolic dysfunction was seen in 34% (32 of 93). Only 10 patients had both normal coronary and normal cardiac imaging. Coronary artery disease and left ventricular systolic dysfunction were previously unrecognized in 60% (38 of 63) and 84% (27 of 32), respectively, with only 33% (21 of 63) and 19% (6 of 32) on evidence-based treatments. Conclusions: Systematic coronary and cardiac imaging of patients with type 2 myocardial infarction identified coronary artery disease in two-thirds and left ventricular systolic dysfunction in one-third of patients. Unrecognized and untreated coronary or cardiac disease is seen in most patients with type 2 myocardial infarction, presenting opportunities for initiation of evidence-based treatments with major potential to improve clinical outcomes. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03338504.

In situ hybridization (ISH) is a technique for the detection of the location of RNA within a tissue of interest. This process uses oligonucleotides with complementary sequences to bind to the target RNA, and colorimetric detection to allow for the visualization of this binding. The process of ISH means that the specific location of the RNA in question can be detected, including in which cell types it is present, and the intracellular location. In the case of long noncoding RNA (lncRNA), which do not lead to the production of proteins, ISH is essential for tissue localization. Moreover, RNA abundance is often lower than for protein-coding genes, thus necessitating enhanced detection through double-digoxigenin (DIG) labeling of the probes. Here, we describe the theory and practicalities of performing ISH for lncRNA, with particular reference to vascular tissues.

A MultiStage Fatigue (MSF) model that admits different hierarchical microstructural features and their stereological information is used to predict the fatigue behavior of 17 different processed aluminum alloys: A000 series (A319, A356, A357, and A380), 2000 series (2024, 2055, 2099, 2198, 2297), 5000 series (5052, 5456), and 7000 series (7050, 7055, 7065, 7075, 7085, 7175). A single set of MSF model constants was validated for all of the aforementioned aluminum alloys, wherein the variation in fatigue life has been captured according to distinct microstructural features (pore size, pore nearest neighbor distance, porosity, particle size, grain size, crystallographic orientation, and misorientation) that differ arising from their native material processing method (casting, rolling, or extrusion). The MSF model’s total number of cycles distinguishes two different regimes: crack incubation (Inc) and Microstructurally Small Crack (long cracks are not considered herein). The previous MSF model in the literature had been associated with the pore size, pore nearest neighbor distance, porosity, particle size, and grain size, but a new contribution in this work is the contribution of the grain orientation and misorientation angles. We show that the MSF model now has the necessary and sufficient equations to predict the Process–Structure–Property relationships for aluminum alloys, allowing for expansion of fatigue prediction even beyond the alloys studied herein.

Supplemental Digital Content is available in the text., Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objectives: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214−/− prevented Ang II-induced periaortic fibrosis, Col1a1, Col3a1, Col5a1, and Tgfb1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214−/− mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214−/− mice. Adoptive transfer of miR-214−/− T cells into RAG1−/− mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214−/−. T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214−/− prevented Ang II-induction of profibrotic T cell cytokines (IL-17, TNF-α, IL-9, and IFN-γ) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T-cell–derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release.

Endothelial cells (ECs) constitute the inner lining of vascular beds in mammals and are crucial for homeostatic regulation of blood vessel physiology, but also play a key role in pathogenesis of many diseases, thereby representing realistic therapeutic targets. However, it has become evident that ECs are heterogeneous, encompassing several subtypes with distinct functions, which makes EC targeting and modulation in diseases challenging. The rise of the new single-cell era has led to an emergence of studies aimed at interrogating transcriptome diversity along the vascular tree, and has revolutionized our understanding of EC heterogeneity from both a physiological and pathophysiological context. Here, we discuss recent landmark studies aimed at teasing apart the heterogeneous nature of ECs. We cover driving (epi)genetic, transcriptomic, and metabolic forces underlying EC heterogeneity in health and disease, as well as current strategies used to combat disease-enriched EC phenotypes, and propose strategies to transcend largely descriptive heterogeneity towards prioritization and functional validation of therapeutically targetable drivers of EC diversity. Lastly, we provide an overview of the most recent advances and hurdles in single EC OMICs. ispartof: CARDIOVASCULAR RESEARCH vol:119 issue:1 pages:6-27 ispartof: location:England status: published

AimsEndothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution.Methods and resultsWe carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature.ConclusionThis study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

A network of molecular factors drives the development, differentiation, and maintenance of endothelial cells. Friend leukemia integration 1 transcription factor (FLI1) is a bona fide marker of endothelial cells during early development. In zebrafish Tg(fli1:EGFP)(y1), we identified two endothelial cell populations, high-fli1(+) and low-fli1(+), by the intensity of green fluorescent protein signal. By comparing RNA-sequencing analysis of non-fli1 expressing cells (fli1(−)) with these two (fli1(+)) cell populations, we identified several up-regulated genes, not previously recognized as important, during endothelial development. Compared with fli1(−) and low-fli1(+) cells, high-fli1(+) cells showed up-regulated expression of the zinc finger transcription factor PRDI-BF1 and RIZ homology domain containing 16 (prdm16). Prdm16 knockdown (KD) by morpholino in the zebrafish larva was associated with impaired angiogenesis and increased number of low-fli1(+) cells at the expense of high-fli1(+) cells. In addition, PRDM16 KD in endothelial cells derived from human-induced pluripotent stem cells impaired their differentiation and migration in vitro. Moreover, zebrafish mutants (mut) with loss of function for the oncogene LIM domain only 2 (lmo2) also showed reduced prdm16 gene expression combined with impaired angiogenesis. Prdm16 expression was reduced further in endothelial (CD31(+)) cells compared with CD31(−) cells isolated from lmo2-mutants (lmo2-mut) embryos. Chromatin immunoprecipitation–PCR demonstrated that Lmo2 binds to the promoter and directly regulates the transcription of prdm16. This work unveils a mechanism by which prdm16 expression is activated in endothelial cells by Lmo2 and highlights a possible therapeutic pathway by which to modulate endothelial cell growth and repair.

Aims Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. Methods and results We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. Conclusion Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells., Graphical Abstract

Arterial calcification is an important hallmark of cardiovascular disease and shares many similarities with skeletal mineralisation. The bone-specific protein osteocalcin (OCN) is an established marker of vascular smooth muscle cell (VSMC) osteochondrogenic trans-differentiation and a known regulator of glucose metabolism. However, the role of OCN in controlling arterial calcification is unclear. We hypothesised that OCN regulates calcification in VSMCs and sought to identify the underpinning signalling pathways. Immunohistochemistry revealed OCN co-localisation with VSMC calcification in human calcified carotid artery plaques. Additionally, 3 mM phosphate treatment stimulated OCN mRNA expression in cultured VSMCs (1.72 fold; p

Aims Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. Methods and results A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. Conclusion A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC., This work was supported by the Medical Research Council [MRC Precision Medicine Doctoral Training Programme to I.R.M. and both the MRC Discovery Award and Programme grant (MC_PC_15075) and MRC Programme: Computational and Disease Genomics (MC_UU_00007/15) to C.P.P.], the Wellcome Trust [Wellcome Trust Senior Research Fellowship in Clinical Science (ref. 103749) to N.C.H.], the European Research Council [Advanced Grant VASCMIR (RE7644) to A.H.B.], and the British Heart Foundation [BHF CVR grant (RM/17/3/ 33381) and BHF Chair of Translational Cardiovascular Sciences to A.H.B.].

Pre-existing immune responses towards adenoviral vector limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest to vectorize novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton-base which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In addition, the vector did not sequester in the liver upon intravenous administration in rodents. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon vaccination. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop safe and efficacious products in the fields of gene therapy and vaccination.IMPORTANCEAdenoviral vectors are currently under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that pre-existing immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrates that we have successfully vectorized a novel adenovirus serotype with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

While humans lack sufficient capacity to undergo cardiac regeneration following injury, zebrafish can fully recover from a range of cardiac insults. Over the past two decades, our understanding of the complexities of both the independent and co-ordinated injury responses by multiple cardiac tissues during zebrafish heart regeneration has increased exponentially. Although cardiomyocyte regeneration forms the cornerstone of the reparative process in the injured zebrafish heart, recent studies have shown that this is dependent on prior neovascularization and lymphangiogenesis, which in turn require epicardial, endocardial, and inflammatory cell signalling within an extracellular milieu that is optimized for regeneration. Indeed, it is the amalgamation of multiple regenerative systems and gene regulatory patterns that drives the much-heralded success of the adult zebrafish response to cardiac injury. Increasing evidence supports the emerging paradigm that developmental transcriptional programmes are re-activated during adult tissue regeneration, including in the heart, and the zebrafish represents an optimal model organism to explore this concept. In this review, we summarize recent advances from the zebrafish cardiovascular research community with novel insight into the mechanisms associated with endogenous cardiovascular repair and regeneration, which may be of benefit to inform future strategies for patients with cardiovascular disease.

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21–32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33–46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states. British Heart Foundation, European Research Council Advanced Grant VASCMIR and British/Israeli Collaborative grant BIRAX.

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

The human transcriptome comprises a complex network of coding and non-coding RNAs implicated in a myriad of biological functions. Non-coding RNAs exhibit highly organized spatial and temporal expression patterns and are emerging as critical regulators of differentiation, homeostasis, and pathological states, including in the cardiovascular system. This review defines the current knowledge gaps, unmet methodological needs, and describes the challenges in dissecting and understanding the role and regulation of the non-coding transcriptome in cardiovascular disease. These challenges include poor annotation of the non-coding genome, determination of the cellular distribution of transcripts, assessment of the role of RNA processing and identification of cell-type specific changes in cardiovascular physiology and disease. We highlight similarities and differences in the hurdles associated with the analysis of the non-coding and protein-coding transcriptomes. In addition, we discuss how the lack of consensus and absence of standardized methods affect reproducibility of data. These shortcomings should be defeated in order to make significant scientific progress and foster the development of clinically applicable non-coding RNA-based therapeutic strategies to lessen the burden of cardiovascular disease. ispartof: CARDIOVASCULAR RESEARCH vol:118 issue:4 pages:1004-1019 ispartof: location:England status: published