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.

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.

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.

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.

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.

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.

Galling Insects of the Brazilian Páramos: Species Richness and Composition Along High-Altitude Grasslands.

In this work, we investigated the factors that determine the distribution of galling insects in high-altitude grasslands, locally called 'campos de altitude' of Mantiqueira Range and tested whether 1) richness of galling insects decreases with altitude, 2) galling insect richness increases with plant richness, 3) variation in galling insect diversity is predominantly a consequence of its ß component, and 4) turnover is the main mechanism driving the beta diversity of both galling insects and plants. Galling insect richness did not exhibit a negative relationship with altitude, but it did increase with plant richness. The additive partition of regional richness (γ) into its local and beta components showed that local diversity (α) of galling insects and plants was relatively low in relation to regional diversity; the ß component incorporated most of the regional diversity. This pattern was also found in the multiscale analysis of the additive partition for galling insects and plants. The beta diversity of galling insects and plants was driven predominantly by the process of turnover and minimally by nesting. The results reported here point out that the spatial distribution of galling insects is best explained by historical factors, such as the distribution of genera and species of key host plants, as well as their relation to habitat, than ecological effects such as hygrothermal stress - here represented by altitude.

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.

Label-Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes.

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label-free microfluidic electrochemical (EC) biosensor with a unique built-in on-chip regeneration capability for continual measurement of cell-secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver-on-a-chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme-linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long-term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

Modelling pulmonary microthrombosis coupled to metastasis: distinct effects of thrombogenesis on tumorigenesis.

Thrombosis can cause localized ischemia and tissue hypoxia, and both of these are linked to cancer metastasis. Vascular micro-occlusion can occur as a result of arrest of circulating tumour cells in small capillaries, giving rise to microthrombotic events that affect flow, creating localized hypoxic regions. To better understand the association between metastasis and thrombotic events, we generated an experimental strategy whereby we modelled the effect of microvascular occlusion in metastatic efficiency by using inert microbeads to obstruct lung microvasculature before, during and after intravenous tumour cell injection. We found that controlled induction of a specific number of these microthrombotic insults in the lungs caused an increase in expression of the hypoxia-inducible transcription factors (HIFs), a pro-angiogenic and pro-tumorigenic environment, as well as an increase in myeloid cell infiltration. Induction of pulmonary microthrombosis prior to introduction of tumour cells to the lungs had no effect on tumorigenic success, but thrombosis at the time of tumour cell seeding increased number and size of tumours in the lung, and this effect was strikingly more pronounced when the micro-occlusion occurred on the day following introduction of tumour cells. The tumorigenic effect of microbead treatment was seen even when thrombosis was induced five days after tumour cell injection. We also found positive correlations between thrombotic factors and expression of HIF2α in human tumours. The model system described here demonstrates the importance of thrombotic insult in metastatic success and can be used to improve understanding of thrombosis-associated tumorigenesis and its treatment.

To PFKFB3 or Not to PFKFB3, That Is the Question.

In this issue of Cancer Cell, Cantelmo et al. describe how reduction of PFKFB3 enzyme activity can promote vascular normalization. The authors show in turn how this affects vascular permeability and can ultimately improve the efficacy of chemotherapeutic agents.