In heart failure reactivation of RNA-binding proteins is associated with the expression of 1,523 fetal-specific isoforms.

Reactivation of fetal-specific genes and isoforms occurs during heart failure. However, the underlying molecular mechanisms and the extent to which the fetal program switch occurs remains unclear. Limitations hindering transcriptome-wide analyses of alternative splicing differences (i.e. isoform switching) in cardiovascular system (CVS) tissues between fetal, healthy adult and heart failure have included both cellular heterogeneity across bulk RNA-seq samples and limited availability of fetal tissue for research. To overcome these limitations, we have deconvoluted the cellular compositions of 996 RNA-seq samples representing heart failure, healthy adult (heart and arteria), and fetal-like (iPSC-derived cardiovascular progenitor cells) CVS tissues. Comparison of the expression profiles revealed that reactivation of fetal-specific RNA-binding proteins (RBPs), and the accompanied re-expression of 1,523 fetal-specific isoforms, contribute to the transcriptome differences between heart failure and healthy adult heart. Of note, isoforms for 20 different RBPs were among those that reverted in heart failure to the fetal-like expression pattern. We determined that, compared with adult-specific isoforms, fetal-specific isoforms encode proteins that tend to have more functions, are more likely to harbor RBP binding sites, have canonical sequences at their splice sites, and contain typical upstream polypyrimidine tracts. Our study suggests that compared with healthy adult, fetal cardiac tissue requires stricter transcriptional regulation, and that during heart failure reversion to this stricter transcriptional regulation occurs. Furthermore, we provide a resource of cardiac developmental stage-specific and heart failure-associated genes and isoforms, which are largely unexplored and can be exploited to investigate novel therapeutics for heart failure.

IFN-γ activates an immune-like regulatory network in the cardiac vascular endothelium.

The regulatory mechanisms underlying the response to pro-inflammatory cytokines during myocarditis are poorly understood. Here, we use iPSC-derived cardiovascular progenitor cells (CVPCs) to model the response to interferon gamma (IFN-γ) during myocarditis. We generate RNA-seq and ATAC-seq for four CVPCs that were treated with IFN-γ and compare them with paired untreated controls. Transcriptional differences after treatment show that IFN-γ initiates an innate immune cell-like response in the vascular cardiac endothelium. IFN-γ treatment also shifts the CVPC transcriptome towards the adult coronary artery and aorta profiles and expands the relative endothelial cell population in all four CVPC lines. Analysis of the accessible chromatin shows that IFN-γ is a potent chromatin remodeler and establishes an IRF-STAT immune-cell like regulatory network. Our findings reveal insights into the endothelial-specific protective mechanisms during myocarditis.

Multi-omic QTL mapping in early developmental tissues reveals phenotypic and temporal complexity of regulatory variants underlying GWAS loci.

Most GWAS loci are presumed to affect gene regulation, however, only ∼43% colocalize with expression quantitative trait loci (eQTLs). To address this colocalization gap, we identify eQTLs, chromatin accessibility QTLs (caQTLs), and histone acetylation QTLs (haQTLs) using molecular samples from three early developmental (EDev) tissues. Through colocalization, we annotate 586 GWAS loci for 17 traits by QTL complexity, QTL phenotype, and QTL temporal specificity. We show that GWAS loci are highly enriched for colocalization with complex QTL modules that affect multiple elements (genes and/or peaks). We also demonstrate that caQTLs and haQTLs capture regulatory variations not associated with eQTLs and explain ∼49% of the functionally annotated GWAS loci. Additionally, we show that EDev-unique QTLs are strongly depleted for colocalizing with GWAS loci. By conducting one of the largest multi-omic QTL studies to date, we demonstrate that many GWAS loci exhibit phenotypic complexity and therefore, are missed by traditional eQTL analyses.

Complex regulatory networks influence pluripotent cell state transitions in human iPSCs.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

Fine mapping spatiotemporal mechanisms of genetic variants underlying cardiac traits and disease.

The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leverage spatiotemporal information on 966 RNA-seq cardiac samples and perform an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identify 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci are enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identifies 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.

eQTL mapping in fetal-like pancreatic progenitor cells reveals early developmental insights into diabetes risk.

The impact of genetic regulatory variation active in early pancreatic development on adult pancreatic disease and traits is not well understood. Here, we generate a panel of 107 fetal-like iPSC-derived pancreatic progenitor cells (iPSC-PPCs) from whole genome-sequenced individuals and identify 4065 genes and 4016 isoforms whose expression and/or alternative splicing are affected by regulatory variation. We integrate eQTLs identified in adult islets and whole pancreas samples, which reveal 1805 eQTL associations that are unique to the fetal-like iPSC-PPCs and 1043 eQTLs that exhibit regulatory plasticity across the fetal-like and adult pancreas tissues. Colocalization with GWAS risk loci for pancreatic diseases and traits show that some putative causal regulatory variants are active only in the fetal-like iPSC-PPCs and likely influence disease by modulating expression of disease-associated genes in early development, while others with regulatory plasticity likely exert their effects in both the fetal and adult pancreas by modulating expression of different disease genes in the two developmental stages.

Analysis of regulatory network modules in hundreds of human stem cell lines reveals complex epigenetic and genetic factors contribute to pluripotency state differences between subpopulations.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discovered 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which were highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlied the coordinated expression of genes in the GNMs. Epigenetic analyses revealed that regulatory networks underlying self-renewal and pluripotency have a surprising level of complexity. Genetic analyses identified thousands of regulatory variants that overlapped predicted transcription factor binding sites and were associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network were significantly enriched for regulatory variants with large effects, suggesting that they may play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work captures the coordinated activity of tens of thousands of regulatory elements in hiPSCs and bins these elements into discrete functionally characterized regulatory networks, shows that regulatory elements in pluripotency networks harbor variants with large effects, and provides a rich resource for future pluripotent stem cell research.

Novel dual inhibitors of vascular endothelial growth factor and VEGFR2 receptor.

BACKGROUND: Vascular endothelial growth factor (VEGF) helps control tumour growth via causing new capillaries growth in tumours. Four cardiac hormones [i.e. vessel dilator, long-acting natriuretic peptide (LANP), kaliuretic peptide (KP) and atrial natriuretic peptide (ANP)] that eliminate up to up to 86% of human small-cell lung cancers growing in mice were investigated for their effects on VEGF and the VEGFR2/KDR/Flk-1 receptor. The VEGFR2 receptor is the main receptor mediating VEGF's cancer-enhancing effects. MATERIALS AND METHODS: Four cardiac hormones were evaluated for their ability to decrease VEGF/VEGFR2 measured by ELISAs in three human cancer cell lines. RESULTS: Vessel dilator, LANP, KP and ANP, over a concentration range of 100 pM to 10 µM, maximally decreased the VEGFR2 receptor in human pancreatic adenocarcinoma cells by 48%, 49%, 74% and 83%. Vessel dilator, LANP, KP and ANP decreased the VEGFR2 receptor by 77%, 89%, 88% and 67% in human small-cell lung cancer cells and by 48%, 92%, 64% and 71% in human prostate cancer cells. These results were confirmed with the cardiac hormones also decreasing the VEGFR2 receptor measured by Western blots. VEGF itself in pancreatic carcinoma cells was decreased by 42%, 58%, 36% and 40% by vessel dilator, LANP, KP and ANP. VEGF levels were decreased 25%, 23%, 17% and 23% in small-cell lung cancer cells and decreased by 24%, 20%, 23% and 24% in prostate cancer cells by vessel dilator, LANP, KP and ANP. CONCLUSION: Four cardiac hormones are the first dual inhibitors of VEGF and the VEGFR2/KDR/Flk-1 receptor.

Potent selective inhibition of STAT 3 versus STAT 1 by cardiac hormones.

Signal transducers and activators of transcription (STATs) are the final "switches" that activate gene expression patterns that lead to human malignancy. Extracellular signal-regulated kinases (ERK 1/2) activate STAT 3; four cardiovascular hormones inhibit ERK 1/2 kinases, leading to the hypothesis that they may also inhibit STATs. These four cardiac hormones, i.e., vessel dilator, long-acting natriuretic peptide (LANP), kaliuretic peptide, and atrial natriuretic peptide (ANP), eliminate human cancers growing in mice. These four cardiac hormones' effects on STATs 1 and 3 were examined in human small-cell lung cancer and human pancreatic adenocarcinoma cells. Vessel dilator, LANP, kaliuretic peptide, and ANP maximally decreased STAT 3 by 88, 54, 55, and 65 %, respectively, at their 1 µM concentrations in human small-cell lung cancer cells and STAT 3 by 66, 57, 70, and 77 % in human pancreatic adenocarcinoma cells, respectively. The cardiac hormones (except LANP) also significantly decreased STAT 3 measured by Western blots. These cardiac hormones did not decrease STAT 1 in either human small-cell lung cancer or pancreatic adenocarcinoma cells. We conclude that these four cardiac hormones are significant inhibitors of STAT 3, but not STAT 1, in human small-cell lung cancer and pancreatic adenocarcinoma cells, which suggests a specificity for these hormones' anticancer mechanism(s) of action enzymology in human cancer cells.

An Anatomic Analysis of Fibula Flap Mandible Reconstructions: Implications for Endosseous Implant Placement.

BACKGROUND: Precise planning and evaluation of the fibula bone are necessary if immediate endosseous implant placement is considered. Limited information is available on the anatomical dimensions or density of fibula used in mandibular reconstructions. This study aimed to describe the morphology and dimensions of the fibula used to reconstruct segmental mandibular defects and contrast the findings with the native mandible. METHODS: A retrospective analysis was performed of patients who underwent segmental mandibulectomy reconstructed with osteocutaneous fibula flaps and had at least one postoperative computed tomography scan. Fibula cross sectional dimensions and densities were evaluated with three-dimensional software. Radiographic measurements were obtained from the contralateral mandible medial to the first molar for comparison. RESULTS: Four hundred seventy-seven fibula cross sections from 159 segments were evaluated. Cross-sectional oval, quadrilateral, triangular, and pentagonal shapes differed significantly in proportion (p < 0.001). Thirty-eight percent of segments (95 percent CI, 30 to 46 percent) had differences in cross-section height greater than 1 mm (p < 0.001). Between segments within the same patient, the median height difference was 1.58 mm (range, 0.14 to 6 mm). The superior cortex density was significantly higher for the fibula than the native mandible; however, the medullary space density was significantly lower (p < 0.001). CONCLUSIONS: The current study comprises the most comprehensive description of fibula morphology in mandibular reconstructions and highlights the significant variability that exists. The findings provide justification for the added time and cost of computer-aided design and computer-aided manufacturing in centers interested in performing immediate dental implant placement, as the technology provides the necessary precision and accuracy.

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues.

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types.

Insights into genetic factors contributing to variability in SARS-CoV-2 susceptibility and COVID-19 disease severity.

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we applied colocalization to compare summary statistics for 16 GWASs from the COVID-19 Host Genetics Initiative to investigate similarities and differences in their genetic signals. We identified 9 loci associated with susceptibility (one with two independent GWAS signals; one with an ethnicity-specific signal), 14 associated with severity (one with two independent GWAS signals; two with ethnicity-specific signals) and one harboring two discrepant GWAS signals (one for susceptibility; one for severity). Utilizing colocalization we also identified 45 GTEx tissues that had eQTL(s) for 18 genes strongly associated with GWAS signals in eleven loci (1-4 genes per locus). Some of these genes showed tissue-specific altered expression and others showed altered expression in up to 41 different tissue types. Our study provides insights into the complex molecular mechanisms underlying inherited predispositions to COVID-19-disease phenotypes.

Th17 reprogramming of T cells in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis (sJIA) begins with fever, rash, and high-grade systemic inflammation but commonly progresses to a persistent afebrile arthritis. The basis for this transition is unknown. To evaluate a role for lymphocyte polarization, we characterized T cells from patients with acute and chronic sJIA using flow cytometry, mass cytometry, and RNA sequencing. Acute and chronic sJIA each featured an expanded population of activated Tregs uncommon in healthy controls or in children with nonsystemic JIA. In acute sJIA, Tregs expressed IL-17A and a gene expression signature reflecting Th17 polarization. In chronic sJIA, the Th17 transcriptional signature was identified in T effector cells (Teffs), although expression of IL-17A at the protein level remained rare. Th17 polarization was abrogated in patients responding to IL-1 blockade. These findings identify evolving Th17 polarization in sJIA that begins in Tregs and progresses to Teffs, likely reflecting the impact of the cytokine milieu and consistent with a biphasic model of disease pathogenesis. The results support T cells as a potential treatment target in sJIA.

Altered monocyte and NK cell phenotypes correlate with posttrauma infection.

BACKGROUND: Trauma induces a complex immune response, requiring a systems biology approach to capture multicellular changes. Using mass cytometry by time-of-flight (CyTOF), we evaluated time-dependent changes in peripheral blood in trauma patients to identify changes correlated with infection. METHODS: Total leukocytes were prepared via red blood cell lysis using peripheral blood samples from trauma patients with an Injury Severity Score greater than 20 at Days 1, 3, and 5 after injury, and from age- and sex-matched uninjured controls. Cells were stained using a 33-marker immunophenotyping CyTOF panel. Statistics were calculated using one-way analysis of variance with multiple comparisons. RESULTS: The CyTOF staining demonstrated changes in many cell subsets. The mean expression intensity of CD86 on monocytes decreased significantly at all time points after injury. When the patients were stratified based on development of infection, there was a trend to decreased CD86 expression on monocytes of those patients that developed subsequent infection. Based on stratification, we identified significantly increased expression of CD39 on NK cells only in patients that developed an infection. CONCLUSION: This study used a systems biology approach to identify novel changes in circulating immune cell subsets in trauma patients correlating with post-traumatic infection. Decreased expression of CD86, a costimulatory molecule, on monocytes demonstrates that trauma affects the innate system's ability to control T-cell immunity. We also found that CD39 expression on NK cells increased significantly in patients with subsequent infection. CD39 is a protein that generates adenosine, which has immunosuppressive effects on several immune cell types including NK cells. In summary, our results point to pathways that may be central to second-hit infections and further study to delineate these pathways could be key to generating clinical biomarkers or targeted immune therapies for trauma patients. LEVEL OF EVIDENCE: Prognostic study, level II.