Lin28A induces energetic switching to glycolytic metabolism in human embryonic kidney cells

Background: Loss of a cell’s capacity to generate sufficient energy for cellular functions is a key hallmark of the ageing process and ultimately leads to a variety of important age-related pathologies such as cancer, Parkinson’s disease and atherosclerosis. Regenerative medicine has sought to reverse these pathologies by reprogramming somatic cells to a more juvenile energetic state using a variety of stem cell factors. One of these factors, Lin28, is considered a candidate for modification in the reprogramming of cellular energetics to ameliorate the ageing process while retaining cell phenotype. Results: Over-expression of Lin28A resulted in key changes to cellular metabolism not observed in wild-type controls. Extracellular pH flux analysis indicated that Lin28A over expression significantly increased the rate of glycolysis, whilst high resolution oxygen respirometry demonstrated a reduced oxygen consumption. Western blot and real-time PCR analysis identified Hexokinase II as one of the key modulators of glycolysis in these cells which was further confirmed by increased glucose transport. A metabolic switching effect was further emphasised by Western blot analysis where the oxygen consuming mitochondrial complex IV was significantly reduced after Lin28A over expression. Conclusions: Results from this study confirm that Lin28A expression promotes metabolic switching to a phenotype that relies predominantly on glycolysis as an energy source, while compromising oxidative phosphorylation. Mechanisms to augment regulated Lin28A in age related pathologies that are characterised by mitochondria dysfunction or in differentiated and aged post-mitotic cells is the future goal of this work

Influence of 2-methoxyestradiol and sex on hypoxia-induced pulmonary hypertension and hypoxia-inducible factor‐1‐α

Background: Women are at greater risk of developing pulmonary arterial hypertension, with estrogen and its downstream metabolites playing a potential role in the pathogenesis of the disease. Hypoxia‐inducible factor‐1‐α (HIF1α) is a pro‐proliferative mediator and may be involved in the development of human pulmonary arterial hypertension. The estrogen metabolite 2‐methoxyestradiol (2ME2) has antiproliferative properties and is also an inhibitor of HIF1α. Here, we examine sex differences in HIF1α signaling in the rat and human pulmonary circulation and determine if 2ME2 can inhibit HIF1α in vivo and in vitro. Methods and Results: HIF1α signaling was assessed in male and female distal human pulmonary artery smooth muscle cells (hPASMCs), and the effects of 2ME2 were also studied in female hPASMCs. The in vivo effects of 2ME2 in the chronic hypoxic rat (male and female) model of pulmonary hypertension were also determined. Basal HIF1α protein expression was higher in female hPASMCs compared with male. Both factor‐inhibiting HIF and prolyl hydroxylase‐2 (hydroxylates HIF leading to proteosomal degradation) protein levels were significantly lower in female hPASMCs when compared with males. In vivo, 2ME2 ablated hypoxia‐induced pulmonary hypertension in male and female rats while decreasing protein expression of HIF1α. 2ME2 reduced proliferation in hPASMCs and reduced basal protein expression of HIF1α. Furthermore, 2ME2 caused apoptosis and significant disruption to the microtubule network. Conclusions: Higher basal HIF1α in female hPASMCs may increase susceptibility to developing pulmonary arterial hypertension. These data also demonstrate that the antiproliferative and therapeutic effects of 2ME2 in pulmonary hypertension may involve inhibition of HIF1α and/or microtubular disruption in PASMCs

Impaired mitochondrial respiration in human carotid plaque atherosclerosis: a potential role for Pink1 in vascular smooth muscle cell energetics

Background and aims: DNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. Methods: Human plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. Results: Human VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMPKinase and hexokinase 2 (Hex2). Conclusions: We have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPKinase and Hexokinase 2

Role of the aryl hydrocarbon receptor in Sugen 5416-induced experimental pulmonary hypertension

Rationale: Rats dosed with the vascular endothelial growth factor (VEGF) inhibitor Sugen 5416 (Su), placed in hypoxia then restored to normoxia has become a widely used model of pulmonary arterial hypertension (PAH). The mechanism by which Su exaccerbates pulmonary hypertension is, however, unclear. Objectives: We investigated Su-activation of the aryl hydrocarbon receptor (AhR) in patient human pulmonary arterial smooth muscle cells (hPASMCs) and patient blood outgrowth endothelial cells (BOECs). We also examined the effect of AhR on aromatase and estrogen levels in the lung. Methods, Measurements and Main Results: Protein and mRNA analysis demonstrated that CYP1A1 was very highly induced in the lungs of Su/hypoxic (Su/Hx) rats. The AhR antagonist CH223191 (8mg/kg/day) reversed the development of PAH in this model in vivo and normalized lung CYP1A1 expression. Increased lung aromatase and estrogen levels in Su/Hx rats were also normalized by CH223191 as was AhR nuclear translocator (ARNT [HIF-1β]) which is shared by HIF-1α and AhR. Su reduced HIF1α expression in hPASMCs. Su induced proliferation in BOECs and increased apoptosis in human pulmonary microvascular endothelial cells (hPMECs) and also induced translocation of AhR to the nucleus in hPASMCs. Under normoxic conditions, hPASMCs do not proliferate to Su. However when grown in hypoxia (1%) Su induced hPASMC proliferation. Conclusion: In combination with hypoxia, Su is proliferative in patient hPASMCs and patient BOECs and Su/Hx-induced PAH in rats may be facilitated by AhR-induced CYP1A1, ARNT and aromatase. Inhibition of the AhR receptor may be a novel approach to the treatment of pulmonary hypertension

Inducing energetic switching using Klotho improves vascular smooth muscle cell phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival

A hierarchical and modular approach to the discovery of robust associations in genome-wide association studies from pooled DNA samples

[Background] One of the challenges of the analysis of pooling-based genome wide association studies is to identify authentic associations among potentially thousands of false positive associations. [Results] We present a hierarchical and modular approach to the analysis of genome wide genotype data that incorporates quality control, linkage disequilibrium, physical distance and gene ontology to identify authentic associations among those found by statistical association tests. The method is developed for the allelic association analysis of pooled DNA samples, but it can be easily generalized to the analysis of individually genotyped samples. We evaluate the approach using data sets from diverse genome wide association studies including fetal hemoglobin levels in sickle cell anemia and a sample of centenarians and show that the approach is highly reproducible and allows for discovery at different levels of synthesis. [Conclusion] Results from the integration of Bayesian tests and other machine learning techniques with linkage disequilibrium data suggest that we do not need to use too stringent thresholds to reduce the number of false positive associations. This method yields increased power even with relatively small samples. In fact, our evaluation shows that the method can reach almost 70% sensitivity with samples of only 100 subjects.Supported by NHLBI grants R21 HL080463 (PS); R01 HL68970 (MHS); K-24, AG025727 (TP); K23 AG026754 (D.T.)

Prevalence of Influenza A viruses in wild migratory birds in Alaska: Patterns of variation in detection at a crossroads of intercontinental flyways

<p>Abstract</p> <p>Background</p> <p>The global spread of the highly pathogenic avian influenza H5N1 virus has stimulated interest in a better understanding of the mechanisms of H5N1 dispersal, including the potential role of migratory birds as carriers. Although wild birds have been found dead during H5N1 outbreaks, evidence suggests that others have survived natural infections, and recent studies have shown several species of ducks capable of surviving experimental inoculations of H5N1 and shedding virus. To investigate the possibility of migratory birds as a means of H5N1 dispersal into North America, we monitored for the virus in a surveillance program based on the risk that wild birds may carry the virus from Asia.</p> <p>Results</p> <p>Of 16,797 birds sampled in Alaska between May 2006 and March 2007, low pathogenic avian influenza viruses were detected in 1.7% by rRT-PCR but no highly pathogenic viruses were found. Our data suggest that prevalence varied among sampling locations, species (highest in waterfowl, lowest in passerines), ages (juveniles higher than adults), sexes (males higher than females), date (highest in autumn), and analytical technique (rRT-PCR prevalence = 1.7%; virus isolation prevalence = 1.5%).</p> <p>Conclusion</p> <p>The prevalence of low pathogenic avian influenza viruses isolated from wild birds depends on biological, temporal, and geographical factors, as well as testing methods. Future studies should control for, or sample across, these sources of variation to allow direct comparison of prevalence rates.</p

Determinants of recovery from post-COVID-19 dyspnoea: analysis of UK prospective cohorts of hospitalised COVID-19 patients and community-based controls

Background The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. Methods We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. Findings We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). Interpretation Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. Funding PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders