Endothelial HIF-2α Suppresses Retinal Angiogenesis in Neonatal Mice by Upregulating NOTCH Signaling.

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia inducible factor (HIF)-α proteins, routing them for polyubiquitination and proteasomal degradation. Typically, HIF-α accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 and Hif-2α. EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIF-α accumulation and VEGF-A upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif-1α, Hif-2α, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF-2α. In PHD2-deficient retinal vasculature, Delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF-2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF-2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

Assessment of Two Restraint Potentials for Coarse-Grained Chemical-Cross-Link-Assisted Modeling of Protein Structures.

The influence of distance restraints from chemical cross-link mass spectroscopy (XL-MS) on the quality of protein structures modeled with the coarse-grained UNRES force field was assessed by using a protocol based on multiplexed replica exchange molecular dynamics, in which both simulated and experimental cross-link restraints were employed, for 23 small proteins. Six cross-links with upper distance boundaries from 4 Å to 12 Å (azido benzoic acid succinimide (ABAS), triazidotriazine (TATA), succinimidyldiazirine (SDA), disuccinimidyl adipate (DSA), disuccinimidyl glutarate (DSG), and disuccinimidyl suberate (BS3)) and two types of restraining potentials ((i) simple flat-bottom Lorentz-like potentials dependent on side chain distance (all cross-links) and (ii) distance- and orientation-dependent potentials determined based on molecular dynamics simulations of model systems (DSA, DSG, BS3, and SDA)) were considered. The Lorentz-like potentials with properly set parameters were found to produce a greater number of higher-quality models compared to unrestrained simulations than the MD-based potentials, because the latter can force too long distances between side chains. Therefore, the flat-bottom Lorentz-like potentials are recommended to represent cross-link restraints. It was also found that significant improvement of model quality upon the introduction of cross-link restraints is obtained when the sum of differences of indices of cross-linked residues exceeds 150.

Japanese urban household carbon footprints during early-stage COVID-19 pandemic were consistent with those over the past decade.

As urbanization accelerates worldwide, substantial energy and services are required to meet the demand from cities, making cities major contributors to adverse environmental consequences. To bridge the knowledge gap in the absence of fine-grained city-level climate protection measures due to data availability and accuracy, this study provides a detailed carbon emission inventory for analyzing the monthly fluctuations based on citizens' daily consumption behaviors. Here, carbon emissions embodied in approximately 500 household consumption items were calculated in 47 prefectural-level cities in Japan from 2011 to June 2021. We analyzed the results considering the regional, seasonal, demand, and emission way-specific aspects, and compared the emission before and during the COVID-19 pandemic. Notably, the carbon footprints during the pandemic were consistent with the previous level despite downtrends in specific categories. This study provides an example of utilizing city-level emission data to improve household green consumption behavior as references for enriching city-level decarbonization paths.

Tailless and hypoxia inducible factor-2α cooperate to sustain proangiogenic states of retinal astrocytes in neonatal mice.

Tailless (TLX, an orphan nuclear receptor) and hypoxia inducible factor-2α (HIF2α) are both essential for retinal astrocyte and vascular development. Tlx-/- mutation and astrocyte specific Hif2α disruption in Hif2αf/f/GFAPCre mice are known to cause defective astrocyte development and block vascular development in neonatal retinas. Here we report that TLX and HIF2α support retinal angiogenesis by cooperatively maintaining retinal astrocytes in their proangiogenic states. While Tlx+/- and Hif2αf/+/GFAPCre mice are phenotypically normal, Tlx+/-/Hif2αf/+/GFAPCre mice display precocious retinal astrocyte differentiation towards non-angiogenic states, along with significantly reduced retinal angiogenesis. In wild-type mice, TLX and HIF2α coexist in the same protein complex, suggesting a cooperative function under physiological conditions. Furthermore, astrocyte specific disruption of Phd2 (prolyl hydroxylase domain protein 2), a manipulation previously shown to cause HIF2α accumulation, did not rescue retinal angiogenesis in Tlx-/- background, which suggests functional dependence of HIF2α on TLX. Finally, the expression of fibronectin and VEGF-A is significantly reduced in retinal astrocytes of neonatal Tlx+/-/Hif2αf/+/GFAPCre mice. Overall, these data indicate that TLX and HIF2α cooperatively support retinal angiogenesis by maintaining angiogenic potential of retinal astrocytes.

Estimating the carbon footprint of household activities in Japan from the time-use perspective.

The household sector is a major driver of energy consumption and greenhouse gas (GHG) emissions. However, most existing studies have only estimated households' carbon footprint from their expenditures. Households' daily activity time, a scarce resource that limits and determines their consumption behavior, has rarely been integrated into the estimation. Incorporating the daily time-use patterns should thus provide a more practical perspective for mitigation policies aiming at promoting sustainable household lifestyles. In this study, by linking household time-use data and expenditure data of Japan, the carbon footprint and the GHG intensity of time of 85 daily household activities constituting the 24 hours in a day are estimated. Compared to the maximal 20-activity disaggregation in existing studies, our detailed 85-category disaggregation of daily time enables unprecedented details on the discrepancies between the carbon footprint from daily activities, many of which have previous been treated as one activity. Results indicate significant carbon mitigation potential in activities with a high GHG intensity of time, such as cooking, bathing, and mobility-related and activities. Average daily GHG emissions were also found to be higher on weekends as time-use patterns shift from paid work to free-time activities, highlighting the need for mitigation strategies on a weekly scale.

Dependence of Retinal Pigment Epithelium Integrity on the NRF2-Heme Oxygenase-1 Axis.

Purpose: Tight junctions (TJs) form the structural basis of retinal pigment epithelium (RPE) barrier functions. Although oxidative stress contributes to age-related macular degeneration, it is unclear how RPE TJ integrity is controlled by redox balance. In this study, we investigated the protective roles of nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor, and heme oxygenase-1 (HO1), a heme-degrading enzyme encoded by the NRF2 target gene HMOX1. Methods: ARPE19 cell cultures and mice, including wild-type, Nrf2-/-, and RPE-specific NRF2-deficient mice, were treated with chemicals that impose oxidative stress or impact heme metabolism. In addition, NRF2 and HO1 expression in ARPE19 cells was knocked down by siRNA. TJ integrity was examined by anti-zonula occludens-1 staining of cultured cells or flatmount RPE tissues from mice. RPE barrier functions were evaluated by transepithelium electrical resistance in ARPE19 cells and immunofluorescence staining for albumin or dextran in eye histological sections. Results: TJ structures and RPE barrier functions were compromised due to oxidant exposure and NRF2 deficiency but were rescued by HO1 inducer. Furthermore, treatment with HO1 inhibitor or heme precursor is destructive to TJ structures and RPE barrier properties. Interestingly, both NRF2 and HO1 were upregulated under oxidative stress, probably as an adaptive response to mitigate oxidant-inflicted damages. Conclusions: Our data indicate that the NRF2-HO1 axis protects TJ integrity and RPE barrier functions by driving heme degradation.

Oxygen-sensing mechanisms in development and tissue repair.

Under normoxia, hypoxia inducible factor (HIF) α subunits are hydroxylated by PHDs (prolyl hydroxylase domain proteins) and subsequently undergo polyubiquitylation and degradation. Normal embryogenesis occurs under hypoxia, which suppresses PHD activities and allows HIFα to stabilize and regulate development. In this Primer, we explain molecular mechanisms of the oxygen-sensing pathway, summarize HIF-regulated downstream events, discuss loss-of-function phenotypes primarily in mouse development, and highlight clinical relevance to angiogenesis and tissue repair.

Monthly direct and indirect greenhouse gases emissions from household consumption in the major Japanese cities.

Urban household consumption contributes substantially to global greenhouse gases (GHGs) emissions. Urban household emissions encompass both direct and indirect emissions, with the former associated with the direct use of fossil fuels and the latter with the emissions embodied in the consumed goods and services. However, there is a lack of consistent and comprehensive datasets outlining in great detail emissions from urban household consumption. To bridge this data gap, we construct an emission inventory of urban household emissions for 52 major cities in Japan that covers around 500 emission categories. The dataset spans from January 2011 to December 2015 and contains 12,384 data records for direct emissions and 1,543,128 records for indirect emissions. Direct emission intensity is provided in g-CO2/JPY to facilitate both future studies of household emission in Japan, as well as act as a reference for the development of detailed household emission inventories in other countries.

Critical role of caveolin-1 in ocular neovascularization and multitargeted antiangiogenic effects of cavtratin via JNK.

Ocular neovascularization is a devastating pathology of numerous ocular diseases and is a major cause of blindness. Caveolin-1 (Cav-1) plays important roles in the vascular system. However, little is known regarding its function and mechanisms in ocular neovascularization. Here, using comprehensive model systems and a cell permeable peptide of Cav-1, cavtratin, we show that Cav-1 is a critical player in ocular neovascularization. The genetic deletion of Cav-1 exacerbated and cavtratin administration inhibited choroidal and retinal neovascularization. Importantly, combined administration of cavtratin and anti-VEGF-A inhibited neovascularization more effectively than monotherapy, suggesting the existence of other pathways inhibited by cavtratin in addition to VEGF-A. Indeed, we found that cavtratin suppressed multiple critical components of pathological angiogenesis, including inflammation, permeability, PDGF-B and endothelial nitric oxide synthase expression (eNOS). Mechanistically, we show that cavtratin inhibits CNV and the survival and migration of microglia and macrophages via JNK. Together, our data demonstrate the unique advantages of cavtratin in antiangiogenic therapy to treat neovascular diseases.

PDGF-CC underlies resistance to VEGF-A inhibition and combinatorial targeting of both suppresses pathological angiogenesis more efficiently.

Anti-VEGF-A therapy has proven to be effective for many neovascular diseases. However, drug resistance to anti-VEGF-A treatment can develop. Also, not all patients with neovascular diseases are responsive to anti-VEGF-A treatment. The mechanisms underlying these important issues remain unclear. In this study, using different model systems, we found that inhibition of VEGF-A directly upregulated PDGF-CC and its receptors in multiple cell types in pathological angiogenesis in vitro and in vivo. Importantly, we further revealed that combinatorial targeting of VEGF-A and PDGF-CC suppressed pathological angiogenesis more efficiently than monotherapy. Given the potent angiogenic activity of PDGF-CC, our findings suggest that the development of resistance to anti-VEGF-A treatment may be caused by the compensatory upregulation of PDGF-CC, and combined inhibition of VEGF-A and PDGF-CC may have therapeutic advantages in treating neovascular diseases.

Vasoprotective effect of PDGF-CC mediated by HMOX1 rescues retinal degeneration.

Blood vessel degeneration is critically involved in nearly all types of degenerative diseases. Therefore strategies to enhance blood vessel protection and survival are highly needed. In this study, using different animal models and cultured cells, we show that PDGF-CC is a potent vascular protective and survival factor. PDGF-CC deficiency by genetic deletion exacerbated blood vessel regression/degeneration in various animal models. Importantly, treatment with PDGF-CC protein not only increased the survival of retinal blood vessels in a model of oxygen-induced blood vessel regression but also markedly rescued retinal and blood vessel degeneration in a disease model of retinitis pigmentosa. Mechanistically, we revealed that heme oxygenase-1 (HMOX1) activity is critically required for the vascular protective/survival effect of PDGF-CC, because blockade of HMOX1 completely abolished the protective effect of PDGF-CC in vitro and in vivo. We further found that both PDGF receptors, PDGFR-ß and PDGFR-α, are required for the vasoprotective effect of PDGF-CC. Thus our data show that PDGF-CC plays a pivotal role in maintaining blood vessel survival and may be of therapeutic value in treating various types of degenerative diseases.

Treatment of T1b glottic SCC: laser vs. radiation--a Canadian multicenter study.

OBJECTIVE: To assess the oncological and functional outcomes of T1b squamous cell carcinoma (SCC) of the glottic larynx treated with laser in comparison with radiation. DESIGN: A Canadian multicenter cohort study. SETTING: Three tertiary referral centers for head and neck cancer- Dalhousie University in Halifax, Nova Scotia, Western University in London, Ontario and the University of Manitoba, Winnipeg. METHODS: Patients with T1b glottic SCC who underwent transoral laser resection or radiation as the primary modality of treatment. OUTCOME MEASURES: Oncological outcomes were evaluated using local control, laryngeal preservation, disease free survival and disease specific survival. Voice outcomes were assessed using the Voice Handicap Index-10 (VHI-10). RESULTS: 63 patients met study criteria. 21 were treated with laser and 42 with radiation. Oncologic outcomes at 2 years for laser and radiation demonstrated local control of 95% and 85.9%; laryngeal preservation of 100% and 85.9%; disease free survival of 88.7% and 85.9% and overall survival of 94.1% and 94.8% respectively. VHI-10 data was available for 23/63 patients. During the last follow up visit VHI-10 ranged from 0 to 11 (median 6) in the laser group and 0 to 34 (median 7) in the radiation group. CONCLUSION: T1b SCC of the glottis can be effectively treated with transoral laser microsurgery with oncological outcomes that are at least equivalent to radiation. For patients with VHI scores, voice quality was similar between the two groups. To our knowledge this is the first study directly comparing the oncologic and voice outcomes with laser and radiation for the treatment of glottic cancer involving the anterior commissure.