Metabolic basis to Sherpa altitude adaptation.

The Himalayan Sherpas, a human population of Tibetan descent, are highly adapted to life in the hypobaric hypoxia of high altitude. Mechanisms involving enhanced tissue oxygen delivery in comparison to Lowlander populations have been postulated to play a role in such adaptation. Whether differences in tissue oxygen utilization (i.e., metabolic adaptation) underpin this adaptation is not known, however. We sought to address this issue, applying parallel molecular, biochemical, physiological, and genetic approaches to the study of Sherpas and native Lowlanders, studied before and during exposure to hypobaric hypoxia on a gradual ascent to Mount Everest Base Camp (5,300 m). Compared with Lowlanders, Sherpas demonstrated a lower capacity for fatty acid oxidation in skeletal muscle biopsies, along with enhanced efficiency of oxygen utilization, improved muscle energetics, and protection against oxidative stress. This adaptation appeared to be related, in part, to a putatively advantageous allele for the peroxisome proliferator-activated receptor A (PPARA) gene, which was enriched in the Sherpas compared with the Lowlanders. Our findings suggest that metabolic adaptations underpin human evolution to life at high altitude, and could have an impact upon our understanding of human diseases in which hypoxia is a feature.

HIF2α-arginase axis is essential for the development of pulmonary hypertension.

Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodeling. The hypoxia-inducible transcription factors (HIFs) HIF-1α and HIF-2α are known to contribute to the process of hypoxic pulmonary vascular remodeling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2α is a critical regulator of hypoxia-induced pulmonary arterial hypertension. The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2α deletion. The RVSP of mice lacking HIF-2α in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic WT mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1α exposed to hypoxia. Endothelial HIF-2α deletion also protected mice from hypoxia remodeling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2α, likewise attenuated many of the pathophysiological symptoms associated with hypoxic pulmonary hypertension. We propose a mechanism whereby chronic hypoxia enhances HIF-2α stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2α in regulating the pulmonary vascular response to hypoxia.

Diverse roles of cell-specific hypoxia-inducible factor 1 in cancer-associated hypercoagulation.

Despite the increased risk of thrombosis in cancer patients compared with healthy individuals, mechanisms that regulate cancer-induced hypercoagulation are incompletely understood. The aim of this study was to investigate whether cell-specific hypoxia-inducible factor (HIF) 1α regulates cancer-associated hypercoagulation, using in vitro clotting assays and in vivo cancer models. In mouse lung and mammary tumor cells, hypoxia led to increases in cell adhesion, clotting, and fibrin deposition; these increases were eliminated in HIF1α null cells. Increased levels of HIF1α were also associated with increased tissue factor expression in human breast tumor samples. Conversely, deletion of endothelial (but not myeloid) cell-specific HIF1α doubled pulmonary fibrin deposition, and trebled thrombus formation compared with wildtype littermates in tumor-bearing mice. Our data suggest that tumor and endothelial cell-specific HIF1α may have opposing roles in cancer-associated coagulation and thrombosis. Off-target effects of manipulating the HIF1 axis in cancer patients should be carefully considered when managing thrombotic complications.

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism.

BACKGROUND: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of ß-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. RESULTS: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARß/δ- and PPARα-dependent mechanism. Enhanced PPARß/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. CONCLUSIONS: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Vertebral artery dissection as the underlying cause of ventral spinal epidural haematoma.

Spontaneous spine epidural haematoma is a rare occurrence, with an incidence of 0.1/100 000 inhabitants/year. The anterior location of the haematoma is very uncommon since the dural sac is firmly attached to the posterior longitudinal ligament. Vertebral artery dissection as its underlying cause is an exceptionally rare event, with only two documented cases.This article presents the case of young woman who arrived at the emergency room with a spinal ventral epidural haematoma extending from C2 to T10, caused by a non-traumatic dissecting aneurysm of the right vertebral artery at V2-V3 segment. Since the patient was tetraparetic, she underwent emergent laminectomy, and the vertebral artery dissection was subsequently treated endovascularly with stenting.Vertebral artery dissection with subsequent perivascular haemorrhage is a possible cause of spontaneous spine epidural haematoma, particularly when located ventrally in the cervical and/or high thoracic column. Hence the importance of a thorough investigation of the vertebral artery integrity.

Hemoglobin in large vessel occlusion: Look further than collaterals.

BACKGROUND: Abnormal hemoglobin (Hb) levels lead to poorer outcomes in ischemic stroke, though the mechanisms remain elusive. We aimed to study the role of Hb on imaging and clinical outcomes, namely on collaterals as it is a known mediator of infarct growth. METHODS: Retrospective cohort study of patients with large vessel occlusion ischemic stroke admitted to our center. Demographics, clinical and imaging variables were collected, particularly baseline hemoglobin, presence of anemia and collateral score. Collaterals were scored from 0 to 3 and defined as poor if 0-1. Multivariable analyses were performed for collateral score and clinical outcomes (3-month mortality and good prognosis). RESULTS: We included 811 patients, 215 (26.5 %) with anemia. Patients with anemia were older, had more comorbidities and more severe strokes. Hemoglobin levels and anemia were not associated with collateral score (OR 0.97, 95 % CI 0.89-1.05, p = 0.414 and OR 0.89, 95 % CI 0.64-1.24, p = 0.487, respectively) nor with poor collaterals (OR 0.96, 95 % CI 0.88-1.05, p = 0.398 and OR 0.86, 95 % CI 0.60-1.23, p = 0.406, respectively). Hb levels were associated with 3-month mortality (OR 0.85, 95 % CI 0.76-0.96, p = 0.008). CONCLUSION: Hemoglobin or anemia were not found to be associated with collateral status. Our results raise further questions regarding the pathophysiology of anemia and outcomes in ischemic stroke, highlighting the need for future research.

Infiltration of Tumors Is Regulated by T cell-Intrinsic Nitric Oxide Synthesis.

Nitric oxide (NO) is a signaling molecule produced by NO synthases (NOS1-3) to control processes such as neurotransmission, vascular permeability, and immune function. Although myeloid cell-derived NO has been shown to suppress T-cell responses, the role of NO synthesis in T cells themselves is not well understood. Here, we showed that significant amounts of NO were synthesized in human and murine CD8+ T cells following activation. Tumor growth was significantly accelerated in a T cell-specific, Nos2-null mouse model. Genetic deletion of Nos2 expression in murine T cells altered effector differentiation, reduced tumor infiltration, and inhibited recall responses and adoptive cell transfer function. These data show that endogenous NO production plays a critical role in T cell-mediated tumor immunity.

Protocol to isolate and culture primary mouse feto-placental endothelial cells.

In the mouse, feto-placental endothelial cells (FPEC) line the inner surface of the feto-placental blood vessels located within placental labyrinthine zone and play critical roles in placental development and function. Here, we present a detailed protocol for isolation and culture of primary mouse FPEC, as well as two complementary methods (immunohistochemistry staining and flow cytometry analysis) to assess their purity. These cells are suitable for downstream ex vivo studies to investigate their functional properties, both in normal and pathological contexts. For complete details on the use and execution of this protocol, please refer to Sandovici et al. (2022).

Hyperoxia Reprogrammes Microvascular Endothelial Cell Response to Hypoxia in an Organ-Specific Manner.

Organ function relies on microvascular networks to maintain homeostatic equilibrium, which varies widely in different organs and during different physiological challenges. The endothelium role in this critical process can only be evaluated in physiologically relevant contexts. Comparing the responses to oxygen flux in primary murine microvascular EC (MVEC) obtained from brain and lung tissue reveals that supra-physiological oxygen tensions can compromise MVEC viability. Brain MVEC lose mitochondrial activity and undergo significant alterations in electron transport chain (ETC) composition when cultured under standard, non-physiological atmospheric oxygen levels. While glycolytic capacity of both lung and brain MVEC are unchanged by environmental oxygen, the ability to trigger a metabolic shift when oxygen levels drop is greatly compromised following exposure to hyperoxia. This is particularly striking in MVEC from the brain. This work demonstrates that the unique metabolism and function of organ-specific MVEC (1) can be reprogrammed by external oxygen, (2) that this reprogramming can compromise MVEC survival and, importantly, (3) that ex vivo modelling of endothelial function is significantly affected by culture conditions. It further demonstrates that physiological, metabolic and functional studies performed in non-physiological environments do not represent cell function in situ, and this has serious implications in the interpretation of cell-based pre-clinical models.

The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth.

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.

Life after Cell Death-Survival and Survivorship Following Chemotherapy.

To prevent cancer cells replacing and outnumbering their functional somatic counterparts, the most effective solution is their removal. Classical treatments rely on surgical excision, chemical or physical damage to the cancer cells by conventional interventions such as chemo- and radiotherapy, to eliminate or reduce tumour burden. Cancer treatment has in the last two decades seen the advent of increasingly sophisticated therapeutic regimens aimed at selectively targeting cancer cells whilst sparing the remaining cells from severe loss of viability or function. These include small molecule inhibitors, monoclonal antibodies and a myriad of compounds that affect metabolism, angiogenesis or immunotherapy. Our increased knowledge of specific cancer types, stratified diagnoses, genetic and molecular profiling, and more refined treatment practices have improved overall survival in a significant number of patients. Increased survival, however, has also increased the incidence of associated challenges of chemotherapy-induced morbidity, with some pathologies developing several years after termination of treatment. Long-term care of cancer survivors must therefore become a focus in itself, such that along with prolonging life expectancy, treatments allow for improved quality of life.

A new genotype 2 subcluster identified among GBV-C strains circulating in the Lisbon metropolitan area of Portugal.

The rate of infection by the GBV-C virus was investigated in a group of 214 individuals at high risk of infection with parenterally transmitted viruses, and all living in the Lisbon metropolitan area (Portugal). RNA was extracted from plasma samples, and a fragment of the 5'-UTR was amplified by RT-PCR, disclosing a high prevalence of infection (40.7%). Most probably due to similar modes of viral transmission, the majority of GBV-C (+) individuals were found to be coinfected with HIV and/or HCV. A genomic region covering part of the E1/E2 glycoprotein coding sequence was amplified from approximately half of the GBV-C positive samples (44/87). Phylogenetic analysis of nucleotide sequences showed segregation of Portuguese GBV-C strains with genotype 1 (G1, n = 10) and genotype 2 (G2, n = 24) references. Genotype 1 was significantly associated with the African descent of those infected. Curiously, some of the strains assigned to genotype 2 were shown to form a separate cluster (designated G2*) in both neighbor-joining and Bayesian phylogenetic trees, which was confirmed by multivariate principal coordinate analysis. However, analysis of the distribution of intra- and intergenotype genetic distances support the hypothesis that rather than corresponding to a new viral genotype, G2* is a geographical subcluster within the genotype 2 radiation. J. Med. Virol. 82:452-459, 2010. (c) 2010 Wiley-Liss, Inc.

Endothelial cells and organ function: applications and implications of understanding unique and reciprocal remodelling.

The microvasculature is a heterogeneous, dynamic and versatile component of the systemic circulation, with a unique ability to locally self-regulate and to respond to organ demand and environmental stimuli. Endothelial cells from different organs display considerable variation, but it is currently unclear to what extent functional properties of organ-specific endothelial cells are intrinsic, acquired and/or reprogrammable. Vascular function is a fundamental pillar of homeostasis, and dysfunction results in systemic consequences for the organism. Additionally, vascular failure can occur downstream of organ disease or environmental stress, often driving an exacerbation of symptoms and pathologies originally independent of the local circulation. The understanding of the molecular mechanisms underlying endothelial physiology and metabolism holds the promise to inform and improve diagnosis, prognosis and treatment options for a myriad of conditions as unrelated as cancer, neurodegeneration or pulmonary hypertension, and likely everything in between, if we consider that also treatments for such conditions are primarily distributed via the bloodstream. However, studying endothelial function has its challenges: the origin, isolation, culture conditions and preconditioning stimuli make this an extremely variable cell type to study and difficult to source. Animal models exist but are neither trivial to generate, nor necessarily adequately translatable to human disease. In this article, we aim to illustrate the breadth of microvascular functions in different environments, highlighting current and pioneering studies that have advanced our insight into the importance of the integrity of this tissue, as well as the limitations posed by its heterogeneity and plasticity.

Circulating Levels of Epirubicin Cause Endothelial Senescence While Compromising Metabolic Activity and Vascular Function.

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.

Hepatitis delta virus interacts with splicing factor SF3B155 and alters pre-mRNA splicing of cell cycle control genes.

Hepatitis delta virus (HDV) is the agent responsible for the most severe form of human viral hepatitis. The HDV genome consists of a single-stranded circular RNA molecule that encodes for one single protein, the delta antigen. Given its simplicity, HDV must make use of several host cellular proteins to accomplish its life cycle processes, including transcription, replication, post-transcriptional, and post-translational modifications. Consequently, identification of the interactions established between HDV components and host proteins assumes a pivotal interest in the search of novel therapeutic targets. Here, we used the yeast three-hybrid system to screen a human liver cDNA library to identify host proteins that interact with the HDV genomic RNA. One of the identified proteins corresponded to the splicing factor SF3B155, a component of the U2snRNP complex that is essential for the early recognition of 3' splice sites in the pre-mRNAs of human genes. We show that the interaction between the HDV genomic RNA and SF3B155 occurs in vivo and that the expression of HDV promotes changes in splicing of human genes whose alternative splicing is SF3B155-dependent. We further show that expression of HDV triggers alterations in several constitutive and alternative splicing events in the tumor suppressor RBM5 transcript, with consequent reduction of its protein levels. This is the first description that HDV expression promotes changes in the splicing of human genes, and we suggest that the HDV-induced alternative splicing changes, through SF3B155 sequester, may contribute for the early progression to hepatocellular carcinoma characteristic of HDV-infected patients.

Author Correction: Acute and chronic hypoxia differentially predispose lungs for metastases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature.

Whilst there is an extensive body of preclinical nanomedicine research, translation to clinical settings has been slow. Here we present a novel approach to the targeted nanoparticle (NP) concept: utilizing both a novel targeting ligand, VNAR (Variable New Antigen Receptor), a shark-derived single chain binding domain, and an under-investigated target in delta-like ligand 4 (DLL4). We describe the development of an anti-DLL4 VNAR and the site-specific conjugation of this to poly(lactic-co-glycolic) acid PEGylated NPs using surface maleimide functional groups. These nanoconjugates were shown to specifically bind DLL4 with high affinity and were preferentially internalized by DLL4-expressing pancreatic cancer cell lines and endothelial cells. Furthermore, a distinct anti-angiogenic effect endowed by the anti-DLL4 VNAR was evident in in vitro tubulogenic assays. Taken together these findings highlight the potential of anti-DLL4 targeted polymeric NPs as a novel therapeutic approach in pancreatic cancer.

Acute and chronic hypoxia differentially predispose lungs for metastases.

Oscillations in oxygen levels affect malignant cell growth, survival, and metastasis, but also somatic cell behaviour. In this work, we studied the effect of the differential expression of the two primary hypoxia inducible transcription factor isoforms, HIF-1α and HIF-2α, and pulmonary hypoxia to investigate how the hypoxia response of the vascular endothelium remodels the lung pre-metastatic niche. Molecular responses to acute versus chronic tissue hypoxia have been proposed to involve dynamic HIF stabilization, but the downstream consequences and the extent to which differential lengths of exposure to hypoxia can affect HIF-isoform activation and secondary organ pre-disposition for metastasis is unknown. We used primary pulmonary endothelial cells and mouse models with pulmonary endothelium-specific deletion of HIF-1α or HIF-2α, to characterise their roles in vascular integrity, inflammation and metastatic take after acute and chronic hypoxia. We found that acute hypoxic response results in increased lung metastatic tumours, caused by HIF-1α-dependent endothelial cell death and increased microvascular permeability, in turn facilitating extravasation. This is potentiated by the recruitment and retention of specific myeloid cells that further support a pro-metastatic environment. We also found that chronic hypoxia delays tumour growth to levels similar to those seen in normoxia, and in a HIF-2α-specific fashion, correlating with increased endothelial cell viability and vascular integrity. Deletion of endothelial HIF-2α rendered the lung environment more vulnerable to tumour cell seeding and growth. These results demonstrate that the nature of the hypoxic challenge strongly influences the nature of the endothelial cell response, and affects critical parameters of the pulmonary microenvironment, significantly impacting metastatic burden. Additionally, this work establishes endothelial cells as important players in lung remodelling and metastatic progression.

Species turnover drives ß-diversity patterns across multiple spatial scales of plant-galling interactions in mountaintop grasslands.

This study describes differences in species richness and composition of the assemblages of galling insects and their host plants at different spatial scales. Sampling was conducted along altitudinal gradients composed of campos rupestres and campos de altitude of two mountain complexes in southeastern Brazil: Espinhaço Range and Mantiqueira Range. The following hypotheses were tested: i) local and regional richness of host plants and galling insects are positively correlated; ii) beta diversity is the most important component of regional diversity of host plants and galling insects; and iii) Turnover is the main mechanism driving beta diversity of both host plants and galling insects. Local richness of galling insects and host plants increased with increasing regional richness of species, suggesting a pattern of unsaturated communities. The additive partition of regional richness (γ) into local and beta components shows that local richnesses (α) of species of galling insects and host plants are low relative to regional richness; the beta (ß) component incorporates most of the regional richness. The multi-scale analysis of additive partitioning showed similar patterns for galling insects and host plants with the local component (α) incorporated a small part of regional richness. Beta diversity of galling insects and host plants were mainly the result of turnover, with little contribution from nesting. Although the species composition of galling insects and host plant species varied among sample sites, mountains and even mountain ranges, local richness remained relatively low. In this way, the addition of local habitats with different landscapes substantially affects regional richness. Each mountain contributes fundamentally to the composition of regional diversity of galling insects and host plants, and so the design of future conservation strategies should incorporate multiple scales.

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.