Primary endothelial cell–specific regulation of hypoxia‐inducible factor (HIF)‐1 and HIF‐2 and their target gene expression profiles during hypoxia

Bartoszewski, Rafał; Moszyńska, Adrianna; Serocki, Marcin; Cabaj, Aleksandra; Polten, Andreas; Ochocka, Renata; Dell’Italia, Louis J.; Bartoszewska, Sylwia; Króliczewski, Jarosław; Dąbrowski, Michał; Collawn, James F.

doi:10.1096/fj.201802650rr

Cited by 155 publications

(154 citation statements)

References 69 publications

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“…HIF1α levels peaked between 4 and 8 hrs of hypoxia exposure (Fig 1A), consistent with previous observations in microvascular endothelial cells (44). The mRNA expression of HIF1α target genes, vascular endothelial growth factor (VEGF), solute carrier family 2 member 1 (SLC2A1, which encodes glucose transporter 1 (GLUT1)), pyruvate kinase 2 (PKM2), N-myc downstream regulated 1 (NDRG1), and carbonic anhydrase 9 (CA9), also increased in response to hypoxia (Fig 1B) (44,45). As expected, the proliferation of HMEC-1 cells was significantly reduced in hypoxia compared to cells grown under normoxic conditions (S1 Fig A).…”

Section: Resultssupporting

confidence: 92%

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia

Cohen

Geck

Toker

2020

Preprint

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21 The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial 22 cells that comprise the vasculature are poorly connected causing vessels to be leaky and exposing 23 the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are 24 generally inefficient and result in acquired resistance to increased hypoxia due to elimination of 25 the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in 26 tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of 27 endothelial cells in response to hypoxia has been relatively unexplored. Here, we measured polar 28 metabolite levels in microvascular endothelial cells exposed to short-and long-term hypoxia with 29 the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor 30 vasculature and improve drug delivery. Many amino acid-related metabolites were altered by 31 hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-32 methionine metabolism. Additionally, there were significant changes in de novo pyrimidine 33 synthesis as well as glutathione and taurine metabolism. These results provide key insights into 34 the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a 35 foundation for future studies to develop therapies that lead to vessel normalization and more 36 efficient drug delivery. Introduction 38The tumor vasculature is highly disordered with both dense regions of vessels and areas 39 that lack vessels. This leads to inefficient blood flow and delivery of nutrients to a tumor, resulting 40 in a microenvironment that is nutrient-poor and hypoxic (1-3). The endothelial cells that comprise 41 disordered vessels are not tightly connected, resulting in leaky vessels (4,5) and exposing 3 42 endothelial cells to a hypoxic environment. This leakiness also enables tumor cell intravasation 43 and metastasis, and significantly impedes efficient drug delivery to tumors (1,3,6-9). Therefore, a 44 number of therapeutic approaches in cancer treatment have focused on normalizing the disordered 45 tumor vasculature. 46Traditionally, the goal of anti-angiogenic therapies (AATs) was to eradicate the vasculature 47 and deprive tumors of nutrients (10). Many AATs are approved or in clinical trials for treatment 48 of solid tumors as monotherapies or in combination with chemotherapy (9,11-15). However, 49 tumors adapt to the hypoxic microenvironment induced by AATs and develop resistance to therapy 50 (16-22). To overcome these limitations, an alternative approach to targeting the tumor vasculature 51 has been explored, whereby pruning and normalization of the tumor vasculature promotes the 52 highly ordered vasculature found within normal tissues. The resulting increased oxygenation and 53 blood flow in turn allows efficient drug delivery (1,3,23). Normalization of the tumor vasculature 54 has been achieved in several clinical studies f...

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Section: Resultssupporting

confidence: 92%

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia

Cohen

Geck

Toker

2020

Preprint

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“…Interestingly, HIF-3α2 enrichment is not observed at the single HRE required for HIF-2 driven EPO regulation at the liver inducibility element immediately 3′ to EPO [17]. This may imply that HIF-3 does not compete for the binding sites used by HIF-1 and HIF-2 as was recently shown to be true between HIF-1 and HIF-2 despite a set of shared target genes [15,40]. Finally, using the HIST1H2BK gene, we show that HIF-3 may redistribute to non-endogenous HREs upon overexpression and that chromatin occupancy studies should be paired with functional assays to dissect endogenous target genes.…”

Section: Discussionmentioning

confidence: 96%

A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin

Tolonen

Heikkilä

Malinen

et al. 2019

Cell. Mol. Life Sci.

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Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxiainducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

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“…MVEC are key regulators of organ homeostasis, and control perfusion and permeability locally to match organ demand (Augustin and Koh, 2017;. Perception and response to oscillations in oxygen availability, whether as a result of changes in tissue need or environmental supply, are essential aspects of MVEC function (Bartoszewski et al, 2019). In this study, primary MVEC from two continuous endothelial capillary networks were isolated from brain and lung tissue, and their responses to hypoxia compared.…”

Section: Discussionmentioning

confidence: 99%

“…As known key mediators of hypoxia response, activation of the two main HIF-a isoforms was assessed in both MVEC populations, to investigate if discrepancies in the HIF-signalling pathway corresponded to differences in cell viability. HIF-1a expression showed the canonical transient upregulation in both lung and brain MVEC (Bartoszewski et al, 2019;Uchida et al, 2004), although consistently higher levels of this isoform was found in cells from the lung, at all time points ( Figure 1C). HIF-2a protein levels decreased only at later time points for lMVEC, but bMVEC showed strikingly higher levels of HIF-2a protein throughout the hypoxia time course ( Figure 1D).…”

Section: Brain and Lung Microvascular Endothelial Cells Respond Diffementioning

confidence: 92%

Microvascular Endothelial Cell Adaptation to Hypoxia Is Organ-Specific and Conditioned by Environmental Oxygen

Reiterer

Eakin

Burke

et al. 2020

Preprint

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Microvascular endothelial cells (MVEC) are plastic, versatile and highly responsive cells, with morphological and functional aspects that uniquely match the tissues they supply. The response of these cells to oxygen oscillations is an essential aspect of tissue homeostasis, and is finely tuned to maintain organ function during physiological and metabolic challenges. Primary MVEC from two continuous capillary networks with distinct organ microenvironments, those of the lung and brain, were pre-conditioned at normal atmospheric (~ 21 %) and physiological (5 and 10 %) O2 levels, and subsequently used to compare organ-specific MVEC hypoxia response. Brain MVEC preferentially stabilise HIF-2α in response to hypoxia, whereas lung MVEC primarily accumulate HIF-1α ; however, this does not result in significant differences at the level of transcriptional activation of hypoxia-induced genes. Glycolytic activity is comparable between brain and lung endothelial cells, and is affected by oxygen pre-conditioning, while glucose uptake is not changed by oxygen pre-conditioning and is observed to be consistently higher in brain MVEC. Conversely, MVEC mitochondrial activity is organ-specific; brain MVEC maintain a higher relative mitochondrial spare capacity at 5% O2, but not following hyperoxic priming. If maintained at supra-physiological O2 levels, both MVEC fail to respond to hypoxia, and have severely compromised and delayed induction of the glycolytic shifts required for survival, an effect which is particularly pronounced in brain MVEC. Oxygen preconditioning also differentially shapes the composition of the mitochondrial electron transport chain (ETC) in the two MVEC populations. Lung MVEC primed at physioxia have lower levels of all ETC complexes compared to hyperoxia, an effect exacerbated by hypoxia. Conversely, brain MVEC expanded in physioxia display increased complex II (SDH) activity, which is further augmented during hypoxia. SDH activity in brain MVEC primed at 21 % O2 is ablated; upon hypoxia, this results in the accumulation of near-toxic levels of succinate in these cells. Our data suggests that, even though MVEC are primarily glycolytic, mitochondrial integrity in brain MVEC is essential for metabolic responses to hypoxia; these responses are compromised when cells are exposed to supra-physiological levels of oxygen. This work demonstrates that the study of MVEC in normal cell culture environments do not adequately represent physiological parameters found in situ, and show that the unique metabolism and function of organ-specific MVEC can be reprogrammed by external oxygen, significantly affecting the timing and degree of downstream responses.

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Primary endothelial cell–specific regulation of hypoxia‐inducible factor (HIF)‐1 and HIF‐2 and their target gene expression profiles during hypoxia

Cited by 155 publications

References 69 publications

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia

A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin

Microvascular Endothelial Cell Adaptation to Hypoxia Is Organ-Specific and Conditioned by Environmental Oxygen

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