Metabolic Responses to Redox Stress in Vascular Cells.

Significance: Redox stress underlies numerous vascular disease mechanisms. Metabolic adaptability is essential for vascular cells to preserve energy and redox homeostasis. Recent Advances: Single-cell technologies and multiomic studies demonstrate significant metabolic heterogeneity among vascular cells in health and disease. Increasing evidence shows that reductive or oxidative stress can induce metabolic reprogramming of vascular cells. A recent example is intracellular L-2-hydroxyglutarate accumulation in response to hypoxic reductive stress, which attenuates the glucose flux through glycolysis and mitochondrial respiration in pulmonary vascular cells and provides protection against further reductive stress. Critical Issues: Regulation of cellular redox homeostasis is highly compartmentalized and complex. Vascular cells rely on multiple metabolic pathways, but the precise connectivity among these pathways and their regulatory mechanisms is only partially defined. There is also a critical need to understand better the cross-regulatory mechanisms between the redox system and metabolic pathways as perturbations in either systems or their cross talk can be detrimental. Future Directions: Future studies are needed to define further how multiple metabolic pathways are wired in vascular cells individually and as a network of closely intertwined processes given that a perturbation in one metabolic compartment often affects others. There also needs to be a comprehensive understanding of how different types of redox perturbations are sensed by and regulate different cellular metabolic pathways with specific attention to subcellular compartmentalization. Lastly, integration of dynamic changes occurring in multiple metabolic pathways and their cross talk with the redox system is an important goal in this multiomics era.

Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

BACKGROUND: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. METHODS: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. RESULTS: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1ß sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. CONCLUSIONS: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

Network medicine in Cardiovascular Research.

The ability to generate multi-omics data coupled with deeply characterizing the clinical phenotype of individual patients promises to improve understanding of complex cardiovascular pathobiology. There remains an important disconnection between the magnitude and granularity of these data and our ability to improve phenotype-genotype correlations for complex cardiovascular diseases. This shortcoming may be due to limitations associated with traditional reductionist analytical methods, which tend to emphasize a single molecular event in the pathogenesis of diseases more aptly characterized by crosstalk between overlapping molecular pathways. Network medicine is a rapidly growing discipline that considers diseases as the consequences of perturbed interactions between multiple interconnected biological components. This powerful integrative approach has enabled a number of important discoveries in complex disease mechanisms. In this review, we introduce the basic concepts of network medicine and highlight specific examples by which this approach has accelerated cardiovascular research. We also review how network medicine is well-positioned to promote rational drug design for patients with cardiovascular diseases, with particular emphasis on advancing precision medicine.

Network Medicine in Pathobiology.

The past decade has witnessed exponential growth in the generation of high-throughput human data across almost all known dimensions of biological systems. The discipline of network medicine has rapidly evolved in parallel, providing an unbiased, comprehensive biological framework through which to interrogate and integrate systematically these large-scale, multi-omic data to enhance our understanding of disease mechanisms and to design drugs that reflect a deep knowledge of molecular pathobiology. In this review, we discuss the key principles of network medicine and the human disease network and explore the latest applications of network medicine in this multi-omic era. We also highlight the current conceptual and technological challenges, which serve as exciting opportunities by which to improve and expand the network-based applications beyond the artificial boundaries of the current state of human pathobiology.

Vital Autotransplantation and Orthodontic Treatment of a Transmigrant Mandibular Canine.

The purpose of this study was to present the case of a 14-year-old patient with an apically closed, transmigrated permanent canine that was autotransplanted into its natural position without endodontic treatment and orthodontically aligned into ideal occlusion. The transplanted canine maintained long-term tooth vitality, physiologic mobility, and normal masticatory function. Also discussed are the clinical considerations and rationale behind this treatment, the clinical procedure, and factors for successully implementing this alternative approach in managing this relatively common developmental dental anomaly.

Defining ELISpot cut-offs from unreplicated test and control wells.

In the absence of replication of wells, empirical criteria for enzyme-linked immunospot (ELISpot) positivity use fixed differences or ratios between spot forming units (SFU) counts between test and control. We propose an alternative approach which first identifies the optimally variance-stabilizing transformation of the SFU counts, based on the Bland-Altman plot of the test and control wells. The second step is to derive a positivity threshold from the difference in between-plate distribution functions of the transformed test and control SFU counts. This method is illustrated using 1309 assay results from a cohort study of influenza in Vietnam in which some, but not all, of the peptide pools have clear tendencies for SFU counts to be higher in test than control wells.

Identification of H5N1-specific T-cell responses in a high-risk cohort in vietnam indicates the existence of potential asymptomatic infections.

BACKGROUND: Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam. METHODS: Peripheral blood mononuclear cells were tested using interferon-γ enzyme-linked immunospot T assays measuring the response to peptides of influenza H5, H3, and H1 hemagglutinin (HA), N1 and N2 neuraminidase, and the internal proteins of H3N2. Horse erythrocyte hemagglutination inhibition assay was performed to detect antibodies against H5N1. RESULTS: Twenty-four of 747 individuals demonstrated H5-specific T-cell responses but little or no cross-reactivity with H3 or H1 HA peptides. H5N1 peptide-specific T-cell lines that did not cross-react with H1 or H3 influenza virus HA peptides were generated. Four individuals also had antibodies against H5N1. CONCLUSIONS: This is the first report of ex vivo H5 HA-specific T-cell responses in a healthy but H5N1-exposed population. Our results indicate that the presence of H5N1-specific T cells could be an additional diagnostic tool for asymptomatic H5N1 infection.

Mouse transmembrane BAX inhibitor motif 3 (Tmbim3) encodes a 38 kDa transmembrane protein expressed in the central nervous system.

Cell survival proteins play an important role throughout nervous system development, normal physiological processes, and pathological conditions. Transmembrane BAX inhibitor motif 3 (TMBIM3, also known as GRINA), is a member of a family of proteins that contain a conserved BAX inhibitor-1 motif. This family of proteins includes several members that have been shown to protect cells from apoptosis. In this study, the authors show that TMBIM3 is expressed in the brain including high levels in the hippocampus. Biochemical and sequence analysis of TMBIM3 demonstrates that the rat, murine, and human genes encode an approximately 38 kDa protein with a predicted seven transmembrane domain topology. A Tmbim3 knockout mouse line did not have an obvious phenotype, but may prove useful in future studies of this family of proteins.

Invariant NKT cells reduce the immunosuppressive activity of influenza A virus-induced myeloid-derived suppressor cells in mice and humans.

Infection with influenza A virus (IAV) presents a substantial threat to public health worldwide, with young, elderly, and immunodeficient individuals being particularly susceptible. Inflammatory responses play an important role in the fatal outcome of IAV infection, but the mechanism remains unclear. We demonstrate here that the absence of invariant NKT (iNKT) cells in mice during IAV infection resulted in the expansion of myeloid-derived suppressor cells (MDSCs), which suppressed IAV-specific immune responses through the expression of both arginase and NOS, resulting in high IAV titer and increased mortality. Adoptive transfer of iNKT cells abolished the suppressive activity of MDSCs, restored IAV-specific immune responses, reduced IAV titer, and increased survival rate. The crosstalk between iNKT and MDSCs was CD1d- and CD40-dependent. Furthermore, IAV infection and exposure to TLR agonists relieved the suppressive activity of MDSCs. Finally, we extended these results to humans by demonstrating the presence of myeloid cells with suppressive activity in the PBLs of individuals infected with IAV and showed that their suppressive activity is substantially reduced by iNKT cell activation. These findings identify what we believe to be a novel immunomodulatory role of iNKT cells, which we suggest could be harnessed to abolish the immunosuppressive activity of MDSCs during IAV infection.

Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals.

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.