Cristina Branco

HIF2α–arginase axis is essential for the development of pulmonary hypertension

Significance The expression of hypoxia-inducible factor (HIF)-2α in pulmonary endothelium of mice influences pulmonary vascular resistance and development of hypoxic pulmonary hypertension (PH) via an arginase-1–dependent mechanism. The HIF-2α:arginase-1 axis influences the homeostatic regulation of nitric oxide synthesis in the lung. Impaired generation of this vasoactive agent contributes to the initial development and vascular remodeling process of PH. 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.

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

BackgroundInsulin 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 β-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.ResultsHerein, 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 PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/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.ConclusionsNitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Metabolic basis to Sherpa altitude adaptation

Significance A relative fall in tissue oxygen levels (hypoxia) is a common feature of many human diseases, including heart failure, lung diseases, anemia, and many cancers, and can compromise normal cellular function. Hypoxia also occurs in healthy humans at high altitude due to low barometric pressures. Human populations resident at high altitude in the Himalayas have evolved mechanisms that allow them to survive and perform, including adaptations that preserve oxygen delivery to the tissues. Here, we studied one such population, the Sherpas, and found metabolic adaptations, underpinned by genetic differences, that allow their tissues to use oxygen more efficiently, thereby conserving muscle energy levels at high altitude, and possibly contributing to the superior performance of elite climbing Sherpas at extreme altitudes. 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.

Autocrine VEGF Isoforms Differentially Regulate Endothelial Cell Behavior

Vascular endothelial growth factor A (VEGF) is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration, and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2). We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO) synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell homeostasis in normoxia and 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.

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

Summary 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.

S110 Hif2a deletion in the pulmonary endothelium prevents hypoxia-induced pulmonary hypertension

Pulmonary arterial hypertension is a progressive and irreversible disease that leads eventually to right heart failure and death. The pathogenesis of this condition involves proliferation of endothelial and smooth muscle cells resulting in vascular remodelling of the pulmonary arterioles. Several factors are implicated in the remodelling process driven by hypoxia including stabilisation of hypoxia-inducible transcription factors (HIFs), HIF1α and HIF2α. Previous studies have shown that heterozygous deletions of HIF1α or HIF2α partially attenuate many of the remodelling process associated with the development of PAH. Consistent with these observations we have found that pulmonary endothelial specific deletion of HIF2α, achieved using murine cre-loxp technologies (L1 or alk1-cre), offers protection against hypoxia-induced PAH. The rise in pulmonary artery pressure (PAP) normally observed following chronic hypoxic challenge was absent in mice with pulmonary endothelial HIF2α deletion. The right ventricular systolic pressure of L1cre- HIF2α mice post hypoxic challenge (26.17±1.67 mmHg, n=7) was not significantly different from untreated WT mice (22.48±1.19 mmHg, n=9) and much lower than the hypertensive values seen in WT littermate controls (41.91±1.88 mmHg, n=12, p<0.0001) and L1cre-HIF1α mice (36.25±2.37 mmHg, n=7, p<0.005). Only minimal remodelling was observed in lung sections from L1cre-HIF2α mice reflecting the normal physiological PAPs following chronic hypoxia. We next questioned whether deletion of lung endothelial HIF2α would be sufficient to reduce downstream arginase-1 and −2 gene expression and in turn influence plasma nitrite/nitrate (NO(X)) concentrations, which would be indicative of changes in nitric oxide homeostasis. The expression of both arginase-1 and −2 were significantly reduced in hypoxia-conditioned whole lung samples from L1cre-HIF2α mice relative to WT littermate controls. Plasma NO(X) concentrations were also significantly elevated in the HIF2α mutant mice when compared to plasma from WT control mice. These observations fit a model whereby reduced arginase-1/2 expression leads to increased availability of l-arginine, and in turn increased NO synthesis via NO synthases. These data offer new insights into the role of pulmonary endothelial HIF2α in causing PAH, and offer new therapeutic opportunities for the treatment of this condition.

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

ABSTRACT 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. Summary: Induction of pulmonary microthrombosis by three distinct methods enhances HIF-a expression and tumour formation; increases in tumorigenesis that are induced by these thrombotic insults occur in a time- and mode-dependent manner.

Pulmonary endothelial HIF2

This is the author accepted manuscript. The final version is available from the Proceedings of the National Academy of Sciences (PNAS) via https://doi.org/10.1073/pnas.1602978113

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Thrombosis causes local blood flow restriction and tissue hypoxia, and both of these are associated with tumor cell metastasis. To better understand the regulation of thrombosis-induced metastasis, we created a model incorporating elements of both processes. Controlled induction of pulmonary microthrombosis caused an increase in expression of first hypoxia-inducible factor (HIF)1α, and subsequently HIF2α. Induction of thrombosis before the introduction of tumor cells to venous circulation had no effect on pulmonary tumor number or size; but thrombosis at the time of tumor cell seeding increased number and size of tumors in the lung. Thrombosis on the day after seeding of tumor cells caused an even greater increase in tumor number and size, and this effect persisted until even when thrombosis was induced five days after the introduction of tumor cells into the blood. Experiments on myeloid HIF1α or HIF2α knockout mice demonstrated that loss of either HIF1α or HIF2α eliminated the advantage given to pulmonary tumorigenesis by thrombotic insult. In primary human tumours, markers of thrombosis were positively correlated with expression of the 2 HIFα isoforms. These data demonstrate the importance of microthrombosis in a novel model of metastasis and the essential role of the myeloid cell-specific HIFα response in mediating this process. Citation Format: Colin E. Evans, Asis Palazon, Jingwei Sim, Petros A. Tyrakis, Par-Ola Bendahl, Mattias Belting, Helene Rundqvist, Cristina Branco, Randall S. Johnson. Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A19.