A validated hypoxia-inducible factor (HIF) signature across tissue compartments predicts clinical outcome in human lung fibrosis

We previously reported that oxidative stress drives pseudohypoxic hypoxia-inducible factor (HIF) pathway activation to promote pathogenetic collagen structure-function in human lung fibrosis (Brereton et al., 2022). Here, through bioinformatic studies we investigate HIF pathway activation status in patients with idiopathic pulmonary fibrosis (IPF) and whether this has prognostic significance. Applying a well-established HIF gene expression signature, we classified publicly available datasets into HIF score-high and score-low groups across multiple tissue compartments. TheHIF scores in lung tissue, bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMC) were increased in IPF patients and significantly correlated with an oxidative stress signature consistent with pseudohypoxic HIF pathway activation. A high HIF score in BAL and in PBMC was a strong independent predictor of mortality in multivariate analysis. Thus, a validated HIF gene signature predicts survival across tissue compartments in IPF and merits prospective study as a non-invasive biomarker of lung fibrosis progression.

Is Hypoxic/Altitude Training an Important Topic in the Field of Hypoxia?

Hypoxia is an essential topic in medical or biological sciences. The main aims of the present study were to examine the most important medical articles (i.e., the top 100 most cited) on hypoxia. We examine how the Nobel-prize awarded hypoxia inducible factor (HIF)-pathway discovery in the early 1990s has changed the thematic composition of this body of literature, with a special emphasis on the studies linking hypoxia and cancer. We searched Pubmed for articles with the terms #Hypox, #Altitude, or #Mountain in the title that have been published in biomedical journals and ranked the articles on their number of citations in Web of Science. A second search was performed in all journals for articles related to hypoxia and cancer. Strikingly, only 12 of the top-100 most-cited articles on hypoxia and only 3 articles of the top-100 articles related to cancer were published before 1995. Moreover, only 5 articles from prior 1995 reached 1000 citations, while 27 articles published in 1995 or later were cited more than 1000 times, most of them on the HIF-1 pathway. Eighty percent of the top-100 articles were related to the HIF pathway, while there were no articles on the application of hypoxia either for therapeutic use (i.e., hypoxic conditioning in patients) or for performance enhancement (i.e., altitude training in athletes). In conclusion, the early-1990s discovery of the HIF pathway and of its molecular regulation has shifted the focus of hypoxia research towards molecular mechanisms and consequences of tissue hypoxia, most notably in cancer. The importance of studies focusing on clinical and performance applications of systemic hypoxia is relatively lower.

Non-canonical Function of a Hif-1α Splice Variant Contributes to the Sustained Flight of Locusts

The hypoxia inducible factor (Hif) pathway is functionally conserved across metazoans in modulating cellular adaptations to hypoxia. However, the functions of this pathway under aerobic physiological conditions are rarely investigated. Here, we show that Hif-1α2, a locust Hif-1α isoform, does not induce canonical hypoxic responses but functions as a specific regulator of locust flight, which is a completely aerobic physiological process. Two Hif-1α splice variants were identified in locusts, a ubiquitously expressed Hif-1α1 and a muscle-predominantly expressed Hif- 1α2. Hif-1α1 that induces typical hypoxic responses upon hypoxia exposure, remains inactive during flight. By contrast, the expression of Hif-1α2, which lacks C-terminal transactivation domain, is less sensitive to oxygen-tension but induced extensively by flying. Hif-1α2 sustains flight endurance by promoting glucose oxidation while simultaneously maintaining redox homeostasis by upregulating the production of a reactive oxygen species (ROS) quencher, DJ-1. Overall, this study reveals a novel Hif-mediated mechanism underlying prolonged aerobic physiological activity.

Iron Deficiency in Polycythemia Vera Increases HIF Activity and Transcription of Prothrombotic Genes

Polycythemia vera (PV) is characterized by elevated red cell mass due to gain-of-function mutations of the tyrosine kinase JAK2 gene. Major causes of morbidity and mortality in PV are venous and arterial thromboses. Risk factors of thrombosis in PV are age > 60 years, previous thrombosis history, and leukocytosis due to increased granulocytes (PMID: 31865003). High hematocrit has been considered as a causative factor of thrombosis due to high viscosity; however, this has been challenged. Phlebotomy to maintain hematocrit below 45% is performed by most hematologists to prevent thrombosis. However, in our prospective controlled study, we reported that phlebotomies, but not high hematocrit, are associated with increased risk of thrombosis in Chuvash erythrocytosis (CE). CE is due to a hypomorphic mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs) (PMID 289208), leading to augmented HIFs. We hypothesize that repeated phlebotomies may increase the risk of thrombosis in PV. We reported that HIFs, and some HIFs-regulated thrombotic genes, are upregulated in PV in both granulocytes and platelets, and even more in PV with a thrombosis history (PMID: 32203583). PV patients with mutated JAK2 have lower ferritin, serum iron, transferrin saturation, and increased soluble transferrin receptor levels compared to JAK2-wild type erythrocytosis (PMID: 30042411), indicating that PV patients with JAK2 mutation are more likely to be iron deficiency (ID). Repeated phlebotomies further augment ID, which increases the level of HIF-1 and HIF-2 by inhibiting the principal negative regulator of HIFs, prolyl hydroxylase domain 2 (PHD2) enzyme. PHD2 requires iron as a co-factor (PMID: 18066546). This led us to hypothesize that further increase of HIFs due to phlebotomy-induced ID increases expression of HIF-regulated prothrombotic genes, leading to increased risk of thrombosis in PV. We studied 17 PV with ID, 32 PV without ID, and 19 healthy control and measured their prothrombotic genes' transcript levels in the granulocytes. Of 17 PVs with ID, 9 (56.3 %) had thrombosis while 14/32 PVs without ID (43.8 %) had thrombosis history. We then measured transcript levels of prothrombotic genes, such as F3 (tissue factor), SELP (P-selectin), THBS1 (thrombospondin-1), SERPINE1(plasminogen activator inhibitor-1) and HIF-target genes VEGFA and SLC2A1 (glucose transporter-1). The transcripts of these genes were upregulated in PV with and without ID compared to the controls. However, PV with ID had higher transcripts of these genes compared to PV without ID, except SERPINE1 (Figure 1). These prothrombotic gene transcripts were inversely correlated with ferritin levels and positively correlated with HIF target genes. Thus, ID in PV further augmented HIFs activity and induced expression of prothrombotic genes. In ongoing studies of PV patients serving as their own controls, PV subjects with ID are treated with hydroxyurea to prevent rebound erythrocytosis. The hydroxyurea treatment alone did not normalize high transcripts of prothrombotic and HIF target genes. However, transcripts of these prothrombotic and HIF-target genes decreased after iron correction while their JAK2V617F allele burden did not change. We then measured transcript levels of these prothrombotic genes in the patients with germline mutations augmenting HIF activity including loss of function VHL, gain-of-function EPAS1 (HIF2a), and loss-of-function EGLN1 (PHD2). These patients have high transcript levels of prothrombotic genes due to high HIF activity although they don`t have ID. Transcripts of F3, SELP, VEGFA, and SLC2A1 of the patients with HIF-pathway mutations were increased compared to the controls and compared to PV without ID but similar to the levels in PV with ID. On the other hand, transcript levels of THBS1 and SERPINE1 in the patients with HIF-pathway mutations were higher than the levels seen in the patients with PV with ID. These data indicate that ID in PV augments HIF activity similar, although not identical, to the patients with germline HIF-pathway mutations and may facilitate propensity to thromboses. These data suggest that therapeutic phlebotomy to control high hematocrit in PV and other erythrocytoses has the potential to increase thrombosis risk. In our ongoing studies, transcript levels of these prothrombotic genes are being correlated with their functions and protein levels. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Hypoxia Promotes Metastasis of Gastric Cancer Through ANXA1 Pathway

Background: To determinethe involvement of hypoxia inducible factor1α (HIF) pathway inmetastasis of gastric cancer (GC). Method: It was determined that the expression of HIF and annexin A1 (ANXA1) in human GC (n=76),using immunohis to chemistry, as well as,the involvement of HIF pathway in GC cell migration and invasion in vitro. The correlation between HIF/ANXA1 and the prognosis wasalso evaluated.Results: HIF1αwas substantiallyupregulatedin GC tissues, compared to that of non-cancer. HIF1α was positively correlated withlymph node metastasis, but inversely correlated with survival in GC patients. HIF1αwas an independent prognostic factor in GC patients, and the combination of HIF1α and ANXA1 might serve asuseful prognostic markers in GCpatients.Furthermore,HIF-1αseems topromote GC migration and invasion through HIF1α/ANXA1/MMP-2 pathway. Conclusions: Our findings suggestthat HIF-1αmay serve as a promising prognostic biomarkerand therapeutic target for inhibiting GC metastasis.

Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells

Background Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxiainducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. Methods We investigated HIF's effect on ENaC expression in mpkCCDcl4 cells (a model of collecting duct principal cells) using real-time PCR and Western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. Results In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of βENaC and γENaC, as well as of Na,K-ATPase. HIF1α silencing increased βENaC and γENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γENaC abundance under high sodium intake. Conclusions This study reveals that γENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

The evolutionary and physiological significance of the Hif pathway in teleost fishes

ABSTRACT The hypoxia-inducible factor (HIF) pathway is a key regulator of cellular O2 homeostasis and an important orchestrator of the physiological responses to hypoxia (low O2) in vertebrates. Fish can be exposed to significant and frequent changes in environmental O2, and increases in Hif-α (the hypoxia-sensitive subunit of the transcription factor Hif) have been documented in a number of species as a result of a decrease in O2. Here, we discuss the impact of the Hif pathway on the hypoxic response and the contribution to hypoxia tolerance, particularly in fishes of the cyprinid lineage, which includes the zebrafish (Danio rerio). The cyprinids are of specific interest because, unlike in most other fishes, duplicated paralogs of the Hif-α isoforms arising from a teleost-specific genome duplication event have been retained. Positive selection has acted on the duplicated paralogs of the Hif-α isoforms in some cyprinid sub-families, pointing to adaptive evolutionary change in the paralogs. Thus, cyprinids are valuable models for exploring the evolutionary significance and physiological impact of the Hif pathway on the hypoxic response. Knockout in zebrafish of either paralog of Hif-1α greatly reduces hypoxia tolerance, indicating the importance of both paralogs to the hypoxic response. Here, with an emphasis on the cardiorespiratory system, we focus on the role of Hif-1α in the hypoxic ventilatory response and the regulation of cardiac function. We explore the effects of the duration of the hypoxic exposure (acute, sustained or intermittent) on the impact of Hif-1α on cardiorespiratory function and compare relevant data with those from mammalian systems.

Loss of Von Hippel–Lindau (VHL) Tumor Suppressor Gene Function: VHL–HIF Pathway and Advances in Treatments for Metastatic Renal Cell Carcinoma (RCC)

Renal cell carcinoma (RCC) is a malignancy of the kidney originating from the tubular epithelium. Inactivation of the von Hippel–Lindau tumor-suppressor gene (VHL) is found in most clear cell renal cell carcinomas (ccRCCs). The VHL–HIF–VEGF/VEGFR pathway, which involves the von Hippel–Lindau tumor suppressor protein (VHL), hypoxia-inducible factor (HIF), vascular endothelial growth factor (VEGF), and its receptor (VEGFR), is a well-studied therapeutic target for metastatic ccRCC. Therefore, over the past decade, anti-angiogenic agents targeting VEGFR have served as the standard treatment for metastatic RCC. Recently, based on the immunomodulatory effect of anti-VEGFR therapy, anti-angiogenic agents and immune checkpoint inhibitor combination strategies have also emerged as therapeutic strategies. These advances were made possible by the improved understanding of the VHL–HIF pathway. In this review, we summarize the historical evolution of ccRCC treatments, with a focus on the involvement of the VHL–HIF pathway.

Regulation of the Hypoxia-Inducible Factor (HIF) by Pro-Inflammatory Cytokines

Hypoxia and inflammation are frequently co-incidental features of the tissue microenvironment in a wide range of inflammatory diseases. While the impact of hypoxia on inflammatory pathways in immune cells has been well characterized, less is known about how inflammatory stimuli such as cytokines impact upon the canonical hypoxia-inducible factor (HIF) pathway, the master regulator of the cellular response to hypoxia. In this review, we discuss what is known about the impact of two major pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), on the regulation of HIF-dependent signaling at sites of inflammation. We report extensive evidence for these cytokines directly impacting upon HIF signaling through the regulation of HIF at transcriptional and post-translational levels. We conclude that multi-level crosstalk between inflammatory and hypoxic signaling pathways plays an important role in shaping the nature and degree of inflammation occurring at hypoxic sites.