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1.
Hum Mol Genet ; 31(7): 1130-1140, 2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-34718575

RESUMO

The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.


Assuntos
Células Endoteliais , Tropomodulina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células Endoteliais/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Pulmão/metabolismo , Camundongos , Tropomodulina/química , Tropomodulina/genética
2.
Int J Mol Sci ; 25(2)2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38255782

RESUMO

Hypoxia not only plays a critical role in multiple disease conditions; it also influences the growth and development of cells, tissues and organs. To identify novel hypoxia-related mechanisms involved in cell and tissue growth, studying a precise hypoxia-sensitive time window can be an effective approach. Drosophila melanogaster has been a useful model organism for studying a variety of conditions, and we focused in this study on the life cycle stages of Drosophila to investigate their hypoxia sensitivity. When normoxia-grown flies were treated with 4% O2 at the pupa stage for 3, 2 and 1 day/s, the eclosion rates were 6.1%, 66.7% and 96.4%, respectively, and, when 4% O2 was kept for the whole pupa stage, this regimen was lethal. Surprisingly, when our hypoxia-adapted flies who normally live in 4% O2 were treated with 4% O2 at the pupa stage, no fly eclosed. Within the pupa stage, the pupae at 2 and 3 days after pupae formation (APF), when treated for 2 days, demonstrated 12.5 ± 8.5% and 23.6 ± 1.6% eclosion, respectively, but this was completely lethal when treated for 3 days. We conclude that pupae, at 2 days APF and for a duration of a minimum of 2 days, were the most sensitive to hypoxia. Our data from our hypoxia-adapted flies clearly indicate that epigenetic factors play a critical role in pupa-stage hypoxia sensitivity.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Pupa , Epigenômica , Hipóxia
3.
Neurobiol Dis ; 174: 105882, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36202289

RESUMO

Early epilepsy is a prominent feature in patients with CDKL5-deficiency disorder (CDD). The underlying mechanism for excessive excitability in CDD is largely unknown. The brain organoid model has been recently developed to resemble many critical features of early human brain development. Here, we used a brain organoid model to investigate the cellular electrophysiological basis for hyper-excitability in CDD patients. Our study employed cortical organoids derived from two CDD patients harboring the same CDKL5 mutation (R59X) and two controls from their healthy parents. Whole-cell patch-clamp recordings revealed higher action potential (AP) firing rate and lower rheobase in both CDD organoids, indicating increased intrinsic neuronal excitability. We further found dysfunction of voltage-gated ion channels in CDD neurons that leads to hyperexcitability, including higher Na+ and K+ current densities and a negative shift in Na+ channel activation. In contrast to neuronal properties, we found that glutamatergic neurotransmission and the electrophysiological properties of glial cells were not altered in CDD organoids. In support of our CDD findings, we further discovered similar electrophysiologic properties in cortical organoids derived from a Rett syndrome (RTT) patient, including alterations in AP firings and Na+ and K+ channel function suggesting a convergent mechanism. Together, our study suggests a critical role of intrinsic neuronal hyperexcitability and ion channel dysfunction, seen in early brain development in both CDD and RTT disorders. This investigation provides potential novel drug targets for developing treatments of early epilepsy in such disorders.


Assuntos
Epilepsia , Células-Tronco Pluripotentes Induzidas , Síndrome de Rett , Humanos , Organoides , Canais Iônicos , Síndrome de Rett/genética , Epilepsia/genética , Proteínas Serina-Treonina Quinases/genética
4.
Mol Psychiatry ; 26(11): 7047-7068, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33888873

RESUMO

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.


Assuntos
Síndromes Epilépticas , Animais , Síndromes Epilépticas/genética , Humanos , Camundongos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases , Proteômica
5.
Int J Mol Sci ; 22(24)2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34948211

RESUMO

Several SLC22 transporters in the human kidney and other tissues are thought to regulate endogenous small antioxidant molecules such as uric acid, ergothioneine, carnitine, and carnitine derivatives. These transporters include those from the organic anion transporter (OAT), OCTN/OCTN-related, and organic cation transporter (OCT) subgroups. In mammals, it has been difficult to show a clear in vivo role for these transporters during oxidative stress. Ubiquitous knockdowns of related Drosophila SLC22s-including transporters homologous to those previously identified by us in mammals such as the "Fly-Like Putative Transporters" FLIPT1 (SLC22A15) and FLIPT2 (SLC22A16)-have shown modest protection against oxidative stress. However, these fly transporters tend to be broadly expressed, and it is unclear if there is an organ in which their expression is critical. Using two tissue-selective knockdown strategies, we were able to demonstrate much greater and longer protection from oxidative stress compared to previous whole fly knockdowns as well as both parent and WT strains (CG6126: p < 0.001, CG4630: p < 0.01, CG16727: p < 0.0001 and CG6006: p < 0.01). Expression in the Malpighian tubule and likely other tissues as well (e.g., gut, fat body, nervous system) appear critical for managing oxidative stress. These four Drosophila SLC22 genes are similar to human SLC22 transporters (CG6126: SLC22A16, CG16727: SLC22A7, CG4630: SLC22A3, and CG6006: SLC22A1, SLC22A2, SLC22A3, SLC22A6, SLC22A7, SLC22A8, SLC22A11, SLC22A12 (URAT1), SLC22A13, SLC22A14)-many of which are highly expressed in the kidney. Consistent with the Remote Sensing and Signaling Theory, this indicates an important in vivo role in the oxidative stress response for multiple SLC22 transporters within the fly renal system, perhaps through interaction with SLC22 counterparts in non-renal tissues. We also note that many of the human relatives are well-known drug transporters. Our work not only indicates the importance of SLC22 transporters in the fly renal system but also sets the stage for in vivo studies by examining their role in mammalian oxidative stress and organ crosstalk.


Assuntos
Drosophila melanogaster/metabolismo , Rim/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/metabolismo , Transporte Biológico/fisiologia , Humanos , Transdução de Sinais/fisiologia
6.
Am J Physiol Regul Integr Comp Physiol ; 318(1): R49-R56, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31617751

RESUMO

Excessive erythrocytosis (EE) is the main sign of chronic mountain sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high-altitude populations worldwide. The pathophysiological mechanism of EE is still controversial, as physiological variability of systemic respiratory, cardiovascular, and hormonal responses to chronic hypoxemia complicates the identification of underlying causes. Induced pluripotent stem cells derived from CMS highlanders showed increased expression of genes relevant to the regulation of erythropoiesis, angiogenesis, cardiovascular, and steroid-hormone function that appear to explain the exaggerated erythropoietic response. However, the cellular response to hypoxia in native CMS cells is yet unknown. This study had three related aims: to determine the hypoxic proliferation of native erythroid progenitor burst-forming unit-erythroid (BFU-E) cells derived from CMS and non-CMS peripheral blood mononuclear cells; to examine their sentrin-specific protease 1 (SENP1), GATA-binding factor 1 (GATA1), erythropoietin (EPO), and EPO receptor (EPOR) expression; and to investigate the functional upstream role of SENP1 in native progenitor differentiation into erythroid precursors. Native CMS BFU-E colonies showed increased proliferation under hypoxic conditions compared with non-CMS cells, together with an upregulated expression of SENP1, GATA1, EPOR; and no difference in EPO expression. Knock-down of the SENP1 gene abolished the augmented proliferative response. Thus, we demonstrate that native CMS progenitor cells produce a larger proportion of erythroid precursors under hypoxia and that SENP1 is essential for proliferation. Our findings suggest a significant intrinsic component for developing EE in CMS highlanders at the cellular and gene expression level that could be further enhanced by systemic factors such as alterations in respiratory control, or differential hormonal patterns.


Assuntos
Doença da Altitude/epidemiologia , Altitude , Células Precursoras Eritroides/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Doença Crônica , Eritropoetina/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Homeostase , Humanos , Hipóxia , Ferro/metabolismo , Leucócitos Mononucleares , Transcriptoma
7.
Int J Mol Sci ; 21(6)2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32183456

RESUMO

The SLC22 family of transporters is widely expressed, evolutionarily conserved, and plays a major role in regulating homeostasis by transporting small organic molecules such as metabolites, signaling molecules, and antioxidants. Analysis of transporters in fruit flies provides a simple yet orthologous platform to study the endogenous function of drug transporters in vivo. Evolutionary analysis of Drosophila melanogaster putative SLC22 orthologs reveals that, while many of the 25 SLC22 fruit fly orthologs do not fall within previously established SLC22 subclades, at least four members appear orthologous to mammalian SLC22 members (SLC22A16:CG6356, SLC22A15:CG7458, CG7442 and SLC22A18:CG3168). We functionally evaluated the role of SLC22 transporters in Drosophila melanogaster by knocking down 14 of these genes. Three putative SLC22 ortholog knockdowns-CG3168, CG6356, and CG7442/SLC22A-did not undergo eclosion and were lethal at the pupa stage, indicating the developmental importance of these genes. Additionally, knocking down four SLC22 members increased resistance to oxidative stress via paraquat testing (CG4630: p < 0.05, CG6006: p < 0.05, CG6126: p < 0.01 and CG16727: p < 0.05). Consistent with recent evidence that SLC22 is central to a Remote Sensing and Signaling Network (RSSN) involved in signaling and metabolism, these phenotypes support a key role for SLC22 in handling reactive oxygen species.


Assuntos
Proteínas de Drosophila , Proteínas de Transporte de Cátions Orgânicos , Estresse Oxidativo , Transdução de Sinais , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo
8.
Am J Physiol Regul Integr Comp Physiol ; 317(6): R763-R769, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618063

RESUMO

Clinical studies have shown that obstructive sleep apnea (OSA) increases atherosclerosis risk. The inflammation, especially mediated by the macrophages via nuclear factor-κB (NF-κB), has been speculated to contribute to atherogenicity in OSA patients. Inhibitor of NF-κB kinase-ß (IKKß) is an essential element of the NF-κB pathway and is linked to atherosclerosis. We previously reported that atherosclerosis was accelerated in pulmonary artery (PA) but not in aorta when low-density lipoprotein receptor knockout (Ldlr-/-) mice were exposed to intermittent hypoxia/hypercapnia (IHH), a surrogate for recurrent upper-airway obstruction. Therefore, we hypothesized that IKKß-dependent NF-κB activation in monocytes and macrophages plays a role in IHH-induced PA atherosclerosis. To test this hypothesis, myeloid restricted IKKß deletion (IkkßΔMye) or control (IkkßF/F) mice were crossed with Ldlr-/- mice to generate double-knockout mice. Then, the mice were exposed to IHH or room air (Air) on high-fat diet for 8 or 16 wk. Lesions of PA and aorta were examined in IkkßΔMye;Ldlr-/- and IkkßF/F;Ldlr-/- male mice under IHH vs. Air. The results revealed that IKKß deletion abolished IHH-induced PA atherosclerosis after 8-wk exposure but not after 16-wk exposure (8 wk: IkkßF/F;Ldlr-/-, IHH 13.5 ± 1.4 vs. Air 5.7 ± 0.7%, P < 0.01; IkkßΔMye;Ldlr-/-, IHH 7.4 ± 1.9% vs. Air 4.6 ± 1.3%, P = 0.24). Both IKKß deletion and IHH had no effects on atherosclerosis in the aorta. Our findings demonstrate that IKKß-dependent NF-κB activity in myeloid-lineage cells plays a critical role in IHH-induced PA atherosclerosis at the early stage.


Assuntos
Aterosclerose/etiologia , Dióxido de Carbono/farmacologia , Hipercapnia/patologia , Hipóxia/patologia , Quinase I-kappa B/metabolismo , Oxigênio/farmacologia , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Colesterol/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases , Artéria Pulmonar/patologia , Receptores de LDL/genética , Aumento de Peso , Quinase Induzida por NF-kappaB
9.
Physiol Genomics ; 50(10): 846-861, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029588

RESUMO

Numerous studies have demonstrated that Na+/H+ exchanger isoform 1 (NHE1) is elevated in myocardial diseases and its effect is detrimental. To better understand the involvement of NHE1, we have previously studied cardiac-specific NHE1 transgenic mice and shown that these mice develop cardiac hypertrophy, interstitial fibrosis, and cardiac dysfunction. The purpose of current study was to identify microRNAs and their mRNA targets involved in NHE1-mediated cardiac injury. An unbiased high-throughput sequencing study was performed on both microRNAs and mRNAs. RNA sequencing showed that differentially expressed genes were enriched in hypertrophic cardiomyopathy pathway by Kyoto Encyclopedia of Genes and Genomes annotation in NHE1 transgenic hearts. These genes were classified as contraction defects (e.g., Myl2, Myh6, Mybpc3, and Actb), impaired intracellular Ca2+ homeostasis (e.g., SERCA2a, Ryr2, Rcan1, and CaMKII delta), and signaling molecules for hypertrophic cardiomyopathy (e.g., Itga/b, IGF-1, Tgfb2/3, and Prkaa1/2). microRNA sequencing revealed that 15 microRNAs were differentially expressed (2-fold, P < 0.05). Six of them (miR-1, miR-208a-3p, miR-199a-5p, miR-21-5p, miR-146a-5p, and miR-30c-5p) were reported to be related to cardiac pathological functions. The integrative analysis of microRNA and RNA sequencing data identified several crucial microRNAs including miR-30c-5p, miR-199a-5p, miR-21-5p, and miR-34a-5p as well as 10 of their mRNA targets that may affect the heart via NFAT hypertrophy and cardiac hypertrophy signaling. Furthermore, important microRNAs and mRNA targets were validated by quantitative PCR. Our study comprehensively characterizes the expression patterns of microRNAs and mRNAs, establishes functional microRNA-mRNA pairs, elucidates the potential signaling pathways, and provides novel insights on the mechanisms underlying NHE1-medicated cardiac injury.


Assuntos
Redes Reguladoras de Genes , MicroRNAs/genética , Miocárdio/metabolismo , RNA Mensageiro/genética , Trocador 1 de Sódio-Hidrogênio/genética , Transcriptoma , Animais , Cardiomegalia/genética , Fibrose/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos Transgênicos , Miocárdio/patologia , Trocador 1 de Sódio-Hidrogênio/metabolismo
10.
Mol Biol Evol ; 34(12): 3154-3168, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29029226

RESUMO

Human high-altitude (HA) adaptation or mal-adaptation is explored to understand the physiology, pathophysiology, and molecular mechanisms that underlie long-term exposure to hypoxia. Here, we report the results of an analysis of the largest whole-genome-sequencing of Chronic Mountain Sickness (CMS) and nonCMS individuals, identified candidate genes and functionally validated these candidates in a genetic model system (Drosophila). We used PreCIOSS algorithm that uses Haplotype Allele Frequency score to separate haplotypes carrying the favored allele from the noncarriers and accordingly, prioritize genes associated with the CMS or nonCMS phenotype. Haplotypes in eleven candidate regions, with SNPs mostly in nonexonic regions, were significantly different between CMS and nonCMS subjects. Closer examination of individual genes in these regions revealed the involvement of previously identified candidates (e.g., SENP1) and also unreported ones SGK3, COPS5, PRDM1, and IFT122 in CMS. Remarkably, in addition to genes like SENP1, SGK3, and COPS5 which are HIF-dependent, our study reveals for the first time HIF-independent gene PRDM1, indicating an involvement of wider, nonHIF pathways in HA adaptation. Finally, we observed that down-regulating orthologs of these genes in Drosophila significantly enhanced their hypoxia tolerance. Taken together, the PreCIOSS algorithm, applied on a large number of genomes, identifies the involvement of both new and previously reported genes in selection sweeps, highlighting the involvement of multiple hypoxia response systems. Since the overwhelming majority of SNPs are in nonexonic (and possibly regulatory) regions, we speculate that adaptation to HA necessitates greater genetic flexibility allowing for transcript variability in response to graded levels of hypoxia.


Assuntos
Aclimatação/genética , Doença da Altitude/genética , Adaptação Fisiológica/genética , Adulto , Alelos , Altitude , Doença da Altitude/metabolismo , Doença da Altitude/fisiopatologia , Animais , Doença Crônica , Drosophila/genética , Evolução Molecular , Frequência do Gene/genética , Haplótipos/genética , Humanos , Hipóxia/genética , Hipóxia/fisiopatologia , Masculino , Peru , Polimorfismo de Nucleotídeo Único/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Sequenciamento Completo do Genoma/métodos
11.
Proc Natl Acad Sci U S A ; 112(33): 10425-30, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26240367

RESUMO

To better understand human adaptation to stress, and in particular to hypoxia, we took advantage of one of nature's experiments at high altitude (HA) and studied Ethiopians, a population that is well-adapted to HA hypoxic stress. Using whole-genome sequencing, we discovered that EDNRB (Endothelin receptor type B) is a candidate gene involved in HA adaptation. To test whether EDNRB plays a critical role in hypoxia tolerance and adaptation, we generated EdnrB knockout mice and found that when EdnrB (-/+) heterozygote mice are treated with lower levels of oxygen (O2), they tolerate various levels of hypoxia (even extreme hypoxia, e.g., 5% O2) very well. For example, they maintain ejection fraction, cardiac contractility, and cardiac output in severe hypoxia. Furthermore, O2 delivery to vital organs was significantly higher and blood lactate was lower in EdnrB (-/+) compared with wild type in hypoxia. Tissue hypoxia in brain, heart, and kidney was lower in EdnrB (-/+) mice as well. These data demonstrate that a lower level of EDNRB significantly improves cardiac performance and tissue perfusion under various levels of hypoxia. Transcriptomic profiling of left ventricles revealed three specific genes [natriuretic peptide type A (Nppa), sarcolipin (Sln), and myosin light polypeptide 4 (Myl4)] that were oppositely expressed (q < 0.05) between EdnrB (-/+) and wild type. Functions related to these gene networks were consistent with a better cardiac contractility and performance. We conclude that EDNRB plays a key role in hypoxia tolerance and that a lower level of EDNRB contributes, at least in part, to HA adaptation in humans.


Assuntos
Coração/fisiologia , Hipóxia/patologia , Receptor de Endotelina B/fisiologia , Aclimatação/genética , Altitude , Animais , Fator Natriurético Atrial/fisiologia , Débito Cardíaco/fisiologia , Etiópia , Feminino , Heterozigoto , Humanos , Ácido Láctico/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/fisiologia , Contração Miocárdica , Cadeias Leves de Miosina/fisiologia , Oxigênio/química , Proteolipídeos/fisiologia , Locos de Características Quantitativas , Receptor de Endotelina B/genética , Análise de Sequência de DNA , Distribuição Tecidual
12.
Am J Respir Cell Mol Biol ; 57(5): 581-588, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28678519

RESUMO

Obstructive sleep apnea (OSA) is a common disorder characterized by intermittent hypoxia and hypercapnia (IHC) during sleep. OSA has been shown to be a risk factor for atherosclerosis, but the relation of IHC to the induction or progression of atherosclerosis is not well understood. To dissect the mechanisms involved, we compared atherosclerotic lesion formation in two mouse models, i.e., apolipoprotein E (ApoE) and low density lipoprotein receptor (Ldlr)-deficient mice, with or without IHC exposure. Ten-week-old ApoE-/- or Ldlr-/- mice were fed a high-fat diet for 4 or 8 weeks while being exposed to IHC for 10 hours/day or room air (RA) for 24 hours/day. En face lesions of the aorta, aortic arch, and pulmonary artery (PA) were examined. Moreover, 3,3-dimethyl-1-butanol (DMB), an inhibitor of microbial trimethylamine (TMA) production, was used to determine the contribution of TMA-oxide (TMAO) to IHC-induced atherosclerosis. Eight weeks of IHC exposure expedited the formation of atherosclerosis in both the PA and aortic arch of ApoE-/- mice, but only in the PA of Ldlr-/- mice (ApoE-/- PA 8 wk, IHC 35.4 ± 1.9% versus RA 8.0 ± 2.8%, P < 0.01). The atherosclerotic lesions evolved faster and to a more severe extent in ApoE-/- mice as compared with Ldlr-/- mice (PA IHC 8 wk, ApoE-/- 35.4 ± 1.9% versus Ldlr-/- 8.2 ± 1.5%, P < 0.01). DMB significantly attenuated but did not totally eliminate IHC-induced PA atherosclerosis. Our findings suggest that IHC, a hallmark of OSA, accelerates the progression of atherosclerosis in the aorta and especially in the PA. This process is partly inhibited by DMB, demonstrating that microbial metabolites may serve as therapeutic targets for OSA-induced atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Hipercapnia/metabolismo , Hipóxia/metabolismo , Metilaminas/metabolismo , Óxidos/metabolismo , Animais , Aterosclerose/genética , Modelos Animais de Doenças , Hipercapnia/complicações , Camundongos Endogâmicos C57BL , Camundongos Knockout , Artéria Pulmonar/metabolismo , Receptores de LDL/metabolismo
13.
J Physiol ; 595(17): 5797-5813, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28688178

RESUMO

KEY POINTS: Changes in gene expression that occur within hours of exposure to hypoxia in in vivo skeletal muscles remain unexplored. Two hours of hypoxia caused significant down-regulation of extracellular matrix genes followed by a shift at 6 h to altered expression of genes associated with the nuclear lumen while respiratory and blood gases were stabilized. Enrichment analysis of mRNAs classified by stability rates suggests an attenuation of post-transcriptional regulation within hours of hypoxic exposure, where PI3K-Akt signalling was suggested to have a nodal role by pathway analysis. Experimental measurements and bioinformatic analyses suggested that the dephosphorylation of Akt after 2 h of hypoxic exposure might deactivate RNA-binding protein BRF1, hence resulting in the selective degradation of mRNAs. ABSTRACT: The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia-tolerant tissues like skeletal muscles. We used RNA-seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down-regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down-regulated genes had mRNA half-lives longer than 2 h, suggesting a role for post-transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K-Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K-Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K-Akt targets, 75% could be explained by a deactivation of adenylate-uridylate-rich element (ARE)-binding protein BRF1, a target of PI3K-Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K-Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the dephosphorylation of ARE-binding protein BRF1.


Assuntos
Hipóxia/genética , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinases/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais
14.
Mol Biol Evol ; 33(2): 501-17, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26576852

RESUMO

The ability to withstand low oxygen (hypoxia tolerance) is a polygenic and mechanistically conserved trait that has important implications for both human health and evolution. However, little is known about the diversity of genetic mechanisms involved in hypoxia adaptation in evolving populations. We used experimental evolution and whole-genome sequencing in Drosophila melanogaster to investigate the role of natural variation in adaptation to hypoxia. Using a generalized linear mixed model we identified significant allele frequency differences between three independently evolved hypoxia-tolerant populations and normoxic control populations for approximately 3,800 single nucleotide polymorphisms. Around 50% of these variants are clustered in 66 distinct genomic regions. These regions contain genes that are differentially expressed between hypoxia-tolerant and normoxic populations and several of the differentially expressed genes are associated with metabolic processes. Additional genes associated with respiratory and open tracheal system development also show evidence of directional selection. RNAi-mediated knockdown of several candidate genes' expression significantly enhanced survival in severe hypoxia. Using genomewide single nucleotide polymorphism data from four high-altitude human populations-Sherpas, Tibetans, Ethiopians, and Andeans, we found that several human orthologs of the genes under selection in flies are also likely under positive selection in all four high-altitude human populations. Thus, our results indicate that selection for hypoxia tolerance can act on standing genetic variation in similar genes and pathways present in organisms diverged by hundreds of millions of years.


Assuntos
Adaptação Biológica/genética , Altitude , Hipóxia/genética , Animais , Evolução Biológica , Drosophila , Frequência do Gene , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único , Seleção Genética
15.
PLoS Genet ; 10(12): e1004844, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25474322

RESUMO

MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/genética , Longevidade/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Envelhecimento/genética , Animais , Animais Geneticamente Modificados , Drosophila/fisiologia , Proteínas de Drosophila/genética , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Estresse Oxidativo/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-23808366

RESUMO

Oxygen is essential for metazoans' life on earth. Oxygen deprivation, or hypoxia, contributes significantly to the pathophysiology of many human diseases. A better understanding of the fundamental molecular and genetic basis for adaptation to low-oxygen environments will help us develop therapeutic strategies to prevent or treat diseases that have hypoxia as a major part of their pathogenesis. Different cells and organisms have evolved different ways to cope with this life-threatening challenge, and the molecular and genetic mechanisms remain largely unknown. The current revolution of genomic technology has advanced our understanding of the genetic basis of many diseases and conditions, including hypoxia tolerance and susceptibility. In this review, we highlight the progress made in understanding the molecular responses to hypoxia in an animal model organism (Drosophila melanogaster) and genetic adaptation to high-altitude hypoxia in humans.


Assuntos
Hipóxia/genética , Oxigênio/metabolismo , Doença da Altitude/genética , Doença da Altitude/fisiopatologia , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Humanos , Hipóxia/fisiopatologia
17.
Am J Hum Genet ; 93(3): 452-62, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23954164

RESUMO

The hypoxic conditions at high altitudes present a challenge for survival, causing pressure for adaptation. Interestingly, many high-altitude denizens (particularly in the Andes) are maladapted, with a condition known as chronic mountain sickness (CMS) or Monge disease. To decode the genetic basis of this disease, we sequenced and compared the whole genomes of 20 Andean subjects (10 with CMS and 10 without). We discovered 11 regions genome-wide with significant differences in haplotype frequencies consistent with selective sweeps. In these regions, two genes (an erythropoiesis regulator, SENP1, and an oncogene, ANP32D) had a higher transcriptional response to hypoxia in individuals with CMS relative to those without. We further found that downregulating the orthologs of these genes in flies dramatically enhanced survival rates under hypoxia, demonstrating that suppression of SENP1 and ANP32D plays an essential role in hypoxia tolerance. Our study provides an unbiased framework to identify and validate the genetic basis of adaptation to high altitudes and identifies potentially targetable mechanisms for CMS treatment.


Assuntos
Doença da Altitude/genética , Genoma Humano/genética , Análise de Sequência de DNA , Adulto , Animais , Doença Crônica , Regulação para Baixo/genética , Drosophila melanogaster/genética , Feminino , Estudos de Associação Genética , Genética Populacional , Genômica , Humanos , Hipóxia/genética , Masculino , Peru , Reprodutibilidade dos Testes , Análise de Sobrevida
18.
BMC Biol ; 13: 101, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26614097

RESUMO

BACKGROUND: Population genetics predicts that tight linkage between new and/or pre-existing beneficial and deleterious alleles should decrease the efficiency of natural selection in finite populations. By decoupling beneficial and deleterious alleles and facilitating the combination of beneficial alleles, recombination accelerates the formation of high-fitness genotypes. This may impose indirect selection for increased recombination. Despite the progress in theoretical understanding, interplay between recombination and selection remains a controversial issue in evolutionary biology. Even less satisfactory is the situation with crossover interference, which is a deviation of double-crossover frequency in a pair of adjacent intervals from the product of recombination rates in the two intervals expected on the assumption of crossover independence. Here, we report substantial changes in recombination and interference in three long-term directional selection experiments with Drosophila melanogaster: for desiccation (~50 generations), hypoxia, and hyperoxia tolerance (>200 generations each). RESULTS: For all three experiments, we found a high interval-specific increase of recombination frequencies in selection lines (up to 40-50% per interval) compared to the control lines. We also discovered a profound effect of selection on interference as expressed by an increased frequency of double crossovers in selection lines. Our results show that changes in interference are not necessarily coupled with increased recombination. CONCLUSIONS: Our results support the theoretical predictions that adaptation to a new environment can promote evolution toward higher recombination. Moreover, this is the first evidence of selection for different recombination-unrelated traits potentially leading, not only to evolution toward increased crossover rates, but also to changes in crossover interference, one of the fundamental features of recombination.


Assuntos
Dessecação , Drosophila melanogaster/fisiologia , Oxigênio/metabolismo , Recombinação Genética , Seleção Genética , Adaptação Fisiológica , Aerobiose , Anaerobiose , Animais , Troca Genética , Drosophila melanogaster/genética
19.
Am J Respir Cell Mol Biol ; 53(3): 355-67, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25569851

RESUMO

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.


Assuntos
Sinalização do Cálcio , Hipertensão Pulmonar/metabolismo , Receptor Notch1/metabolismo , Vasoconstrição , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Hipóxia Celular , Células Cultivadas , Humanos , Hipertensão Pulmonar/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Proteínas Serrate-Jagged , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6
20.
Physiology (Bethesda) ; 29(6): 403-12, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25362634

RESUMO

Chronic mountain sickness (CMS) is a disease that affects many high-altitude dwellers, particularly in the Andean Mountains in South America. The hallmark symptom of CMS is polycythemia, which causes increased risk of pulmonary hypertension and stroke (among other symptoms). A prevailing hypothesis in high-altitude medicine is that CMS results from a population-specific "maladaptation" to the hypoxic conditions at high altitude. In contrast, the prevalence of CMS is very low in other high-altitude populations (e.g., Tibetans and Ethiopians), which are seemingly well adapted to hypoxia. In recent years, concurrent with the advent of genomic technologies, several studies have investigated the genetic basis of adaptation to altitude. These studies have identified several candidate genes that may underlie the adaptation, or maladaptation. Interestingly, some of these genes are targeted by known drugs, raising the possibility of new treatments for CMS and other ischemic diseases. We review recent discoveries, alongside the methodologies used to obtain them, and outline some of the challenges remaining in the field.


Assuntos
Doença da Altitude/genética , Altitude , Hipóxia/fisiopatologia , Aclimatação , Doença da Altitude/etnologia , Doença da Altitude/fisiopatologia , Doença da Altitude/terapia , Etiópia/epidemiologia , Interação Gene-Ambiente , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Hipóxia/etnologia , Hipóxia/genética , Fenótipo , Prevalência , Fatores de Proteção , Fatores de Risco , Tibet/epidemiologia
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