Loss of Hif-2α Rescues the Hif-1α Deletion Phenotype of Neonatal Respiratory Distress In Mice.

Hypoxia is a state of decreased oxygen reaching the tissues of the body. During prenatal development, the fetus experiences localized occurrences of hypoxia that are essential for proper organogenesis and survival. The response to decreased oxygen availability is primarily regulated by hypoxia-inducible factors (HIFs), a family of transcription factors that modulate the expression of key genes involved in glycolysis, angiogenesis, and erythropoiesis. HIF-1α and HIF-2α, two key isoforms, are important in embryonic development, and likely are involved in lung morphogenesis. We have recently shown that the inducible loss of Hif-1α in lung epithelium starting at E4.5 leads to death within an hour of parturition, with symptoms similar to neonatal respiratory distress syndrome (RDS). In addition to Hif-1α, Hif-2α is also expressed in the developing lung, although the overlapping roles of Hif-1α and Hif-2α in this context are not fully understood. To further investigate the independent role of Hif-2α in lung epithelium and its ability to alter Hif-1α-mediated lung maturation, we generated two additional lung-specific inducible Hif-α knockout models (Hif-2α and Hif-1α+Hif-2α). The intrauterine loss of Hif-2α in the lungs does not lead to decreased viability or observable phenotypic changes in the lung. More interestingly, survivability observed after the loss of both Hif-1α and Hif-2α suggests that the loss of Hif-2α is capable of rescuing the neonatal RDS phenotype seen in Hif-1α-deficient pups. Microarray analyses of lung tissue from these three genotypes identified several factors, such as Scd1, Retlnγ, and Il-1r2, which are differentially regulated by the two HIF-α isoforms. Moreover, network analysis suggests that modulation of hormone-mediated, NF-κB, C/EBPα, and c-MYC signaling are central to HIF-mediated changes in lung development.

Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1α contrib utes to cobalt toxicity in vitro, loss of HIF1α in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1α and HIF2α show unique tissue expression profiles, and HIF2α is known to be the predominant HIF mRNA isoform in the adult lung. Thus, if HIF2α activation by cobalt contributes to pathophysiology of HMLD, we hypothesized that loss of HIF2α in lung epithelium would provide protection from cobalt-induced inflammation. Mice with HIF2α-deficiency in Club and alveolar type II epithelial cells (ATIIs) (HIF2α(Δ/Δ)) were exposed to cobalt (60 µg/day) or saline using a subacute occupational exposure model. Bronchoalveolar lavage cellularity, cytokines, qRT-PCR, and histopathology were analyzed. Results show that loss of HIF2α leads to enhanced eosinophilic inflammation and increased goblet cell metaplasia. Additionally, control mice demonstrated a mild recovery from cobalt-induced lung injury compared with HIF2α(Δ/Δ) mice, suggesting a role for epithelial HIF2α in repair mechanisms. The expression of important cytokines, such as interleukin (IL)-5 and IL-10, displayed significant differences following cobalt exposure when HIF2α(Δ/Δ) and control mice were compared. In summary, our data suggest that although loss of HIF2α does not afford protection from cobalt-induced lung inflammation, epithelial HIF2α signaling does play an important role in modulating the inflammatory and repair response in the lung.

Neonatal epithelial hypoxia inducible factor-1α expression regulates the response of the lung to experimental asthma.

Allergic airway disease is characterized by a T helper type 2 cell-mediated airway inflammation and airway hyperresponsiveness. Little is known about the role of hypoxia-mediated signaling in the progression of the disease. To address this knowledge gap, a mouse model was created in which doxycycline exposure induces the functional deletion of hypoxia inducible factor-1α from alveolar type II and Clara cells of the lung. When hypoxia inducible factor-1α deletion was induced during the early postnatal development period of the lung, the mice displayed an enhanced response to the ovalbumin model of allergic airway disease. These hypoxia inducible factor-1α-deficient mice exhibit increased cellular infiltrates, eosinophilia in the lavage fluid and parenchyma, and T helper type 2 cytokines, as compared with ovalbumin-treated control mice. Moreover, these hypoxia inducible factor-1α-deficient mice display increased airway resistance when compared with their control counterparts. Interestingly, if the loss of hypoxia inducible factor-1α was induced in early adulthood, the exacerbated phenotype was not observed. Taken together, these results suggest that epithelial hypoxia inducible factor-1α plays an important role in establishing the innate immunity of the lung and epithelial-specific deficiency in the transcription factor, during early postnatal development, increases the severity of inflammation and functional airway resistance, following ovalbumin challenge. Finally, these results might explain some of the chronic respiratory pathology observed in premature infants, especially those that receive supplemental oxygen. This early hyperoxic exposure, from normal ambient and supplemental oxygen, would presumably inhibit normal hypoxia inducible factor-1α signaling, mimicking the functional deletion described.

The role of hypoxia-inducible factor-1α in acetaminophen hepatotoxicity.

Hypoxia-inducible factor-1α (HIF-1α) is a critical transcription factor that controls oxygen homeostasis in response to hypoxia, inflammation, and oxidative stress. HIF has been implicated in the pathogenesis of liver injury in which these events play a role, including acetaminophen (APAP) overdose, which is the leading cause of acute liver failure in the United States. APAP overdose has been reported to activate HIF-1α in mouse livers and isolated hepatocytes downstream of oxidative stress. HIF-1α signaling controls many factors that contribute to APAP hepatotoxicity, including mitochondrial cell death, inflammation, and hemostasis. Therefore, we tested the hypothesis that HIF-1α contributes to APAP hepatotoxicity. Conditional HIF-1α deletion was generated in mice using an inducible Cre-lox system. Control (HIF-1α-sufficient) mice developed severe liver injury 6 and 24 h after APAP overdose (400 mg/kg). HIF-1α-deficient mice were protected from APAP hepatotoxicity at 6 h, but developed severe liver injury by 24 h, suggesting that HIF-1α is involved in the early stage of APAP toxicity. In further studies, HIF-1α-deficient mice had attenuated thrombin generation and reduced plasminogen activator inhibitor-1 production compared with control mice, indicating that HIF-1α signaling contributes to hemostasis in APAP hepatotoxicity. Finally, HIF-1α-deficient animals had decreased hepatic neutrophil accumulation and plasma concentrations of interleukin-6, keratinocyte chemoattractant, and regulated upon activation normal T cell expressed and secreted compared with control mice, suggesting an altered inflammatory response. HIF-1α contributes to hemostasis, sterile inflammation, and early hepatocellular necrosis during the pathogenesis of APAP toxicity.

Acute cobalt-induced lung injury and the role of hypoxia-inducible factor 1alpha in modulating inflammation.

Air pollution is a critical factor in the development and exacerbation of pulmonary diseases. Ozone, automobile exhaust, cigarette smoke, and metallic dust are among the potentially harmful pollution components that are linked to disease progression. Transition metals, such as cobalt, have been identified at significant levels in air pollution. Cobalt exerts numerous biological effects, including mimicking hypoxia. Similar to hypoxia, cobalt exposure results in the stabilization of hypoxia-inducible factors (HIFs), a family of proteins that regulate the cellular response to oxygen deficit. HIFs also play an important role in innate immunity and inflammatory processes. To characterize the role of HIF1alpha, the most ubiquitously expressed HIF, in the early events during cobalt-induced lung inflammation, an inducible lung-specific HIF1alpha deletion model was employed. Control mice showed classical signs of metal-induced injury following cobalt exposure, including neutrophilic infiltration and induction of Th1 cytokines. In contrast, HIF1alpha-deficient mice exhibited pronounced eosinophil counts in bronchoalveolar lavage fluid and lung tissue complemented with Th2 cytokine induction. The timing of these results suggests that the loss of epithelial-derived HIF1alpha alters the lung's innate immune response and biases the tissue toward a Th2-mediated inflammation.

A simplified approach to human islet quality assessment.

BACKGROUND: The successful treatment of patients with type 1 diabetes by islet transplantation is affected by a multitude of factors of which infusion of the highest quality tissue is essential. The current standard pretransplant quality assessments lack sensitivity, accuracy, and objectivity in the determination of islet viability and potency. We hypothesized that a multiparametric approach focused on islet cell metabolic state, mitochondrial integrity, and in vitro glucose-stimulated insulin secretion (GSIS) could provide data predictive of in vivo function. The objective of this study was to validate a novel set of islet quality assays and develop a simplified islet quality scoring system for both basic research and clinical applications. METHODS: A series of 42 human islet preparations were screened using standard and novel methods, which included determination of yield, viability by fluorescent microscopy, GSIS, percentage of islet loss in culture, quantification of adenine nucleotides, flow cytometric measurement of viability, apoptosis, and mitochondrial membrane potential (MMP). In vivo functional potency was tested by minimal model transplant in streptozotocin-induced diabetic NOD.scid mice. RESULTS: Functionally potent islet preparations showed significantly greater numbers of cells with polarized MMP, higher ATP-to-ADP ratios, and increased glucose-induced insulin secretion. The MMP, ATP-to-ADP ratio, and GSIS data were combined into a single islet scoring formula that showed more than 86% accuracy in predicting in vivo functional potency. CONCLUSIONS: Our study demonstrates that a multiparametric approach using objective assessments focused on islet cell mitochondrial integrity and in vitro function can provide data predictive of in vivo function.

IL-1beta receptor blockade protects islets against pro-inflammatory cytokine induced necrosis and apoptosis.

Pro-inflammatory cytokines (PIC) impair islet viability and function by activating inflammatory pathways that induce both necrosis and apoptosis. The aim of this study was to utilize an in vitro rat islet model to evaluate the efficacy of a clinically approved IL-1 receptor antagonist (Anakinra) in blocking PIC induced islet impairment. Isolated rat islets were cultured for 48 h +/- PIC (IL-1beta, IFNgamma, and TNFalpha) and +/-IL-1ra then assayed for cellular integrity by flow cytometry, MAPK phosphorylation by proteome array, and gene expression by RT-PCR. Nitric oxide (NO) release into the culture media was measured by Griess reaction. Islet functional potency was tested by glucose stimulated insulin secretion (GSIS) and by transplantation into streptozotocin-induced diabetic NOD.scid mice. Rat islets cultured with PIC upregulated genes for NOS2a, COX2, IL6, IL1b, TNFa, and HMOX1. IL-1ra prevented the PIC induced upregulation of all of these genes except for TNFa. Inhibition of PIC induced iNOS by NG-monomethyl-L-arginine (NMMA) only blocked the increased expression of HMOX1. IL-1ra completely abrogated the effects of PIC with respect to NO production, necrosis, apoptosis, mitochondrial dysfunction, GSIS, and in vivo potency. IL-1ra was not effective at preventing the induction of necrosis or apoptosis by exogenous NO. These data demonstrate that Anakinra is an effective agent to inhibit the activation of IL-1beta dependent inflammatory pathways in cultured rat islets and support the extension of its application to human islets in vitro and potentially as a post transplant therapy.