Biomedical subjects

Gwyneth Harry

Publications and source records attributed to Gwyneth Harry.

9 recordsLinked to original sources

PubMed2007

A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth.

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DeltaPsim) and low expression of the K+ channel Kv1.5, both contributing to apoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DeltaPsim, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Animals↗

PubMed2006

An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension.

BACKGROUND: The cause of pulmonary arterial hypertension (PAH) was investigated in humans and fawn hooded rats (FHR), a spontaneously pulmonary hypertensive strain. METHODS AND RESULTS: Serial Doppler echocardiograms and cardiac catheterizations were performed in FHR and FHR/BN1, a consomic control that is genetically identical except for introgression of chromosome 1. PAH began after 20 weeks of age, causing death by &60 weeks. FHR/BN1 did not develop PAH. FHR pulmonary arterial smooth muscle cells (PASMCs) had a rarified reticulum of hyperpolarized mitochondria with reduced expression of electron transport chain components and superoxide dismutase-2. These mitochondrial abnormalities preceded PAH and persisted in culture. Depressed mitochondrial reactive oxygen species (ROS) production caused normoxic activation of hypoxia inducible factor (HIF-1alpha), which then inhibited expression of oxygen-sensitive, voltage-gated K+ channels (eg, Kv1.5). Disruption of this mitochondrial-HIF-Kv pathway impaired oxygen sensing (reducing hypoxic pulmonary vasoconstriction, causing polycythemia), analogous to the pathophysiology of chronically hypoxic Sprague-Dawley rats. Restoring ROS (exogenous H2O2) or blocking HIF-1alpha activation (dominant-negative HIF-1alpha) restored Kv1.5 expression/function. Dichloroacetate, a mitochondrial pyruvate dehydrogenase kinase inhibitor, corrected the mitochondrial-HIF-Kv pathway in FHR-PAH and human PAH PASMCs. Oral dichloroacetate regressed FHR-PAH and polycythemia, increasing survival. Chromosome 1 genes that were dysregulated in FHRs and relevant to the mitochondria-HIF-Kv pathway included HIF-3alpha (an HIF-1alpha repressor), mitochondrial cytochrome c oxidase, and superoxide dismutase-2. Like FHRs, human PAH-PASMCs had dysmorphic, hyperpolarized mitochondria; normoxic HIF-1alpha activation; and reduced expression/activity of HIF-3alpha, cytochrome c oxidase, and superoxide dismutase-2. CONCLUSIONS: FHRs have a chromosome 1 abnormality that disrupts a mitochondria-ROS-HIF-Kv pathway, leading to PAH. Similar abnormalities occur in idiopathic human PAH. This study reveals an intersection between oxygen-sensing mechanisms and PAH. The mitochondria-ROS-HIF-Kv pathway offers new targets for PAH therapy.

Animals↗

PubMed2005

Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization.

BACKGROUND: Bronchopulmonary dysplasia (BPD) and pulmonary emphysema, both significant global health problems, are characterized by a loss of alveoli. Vascular endothelial growth factor (VEGF) is a trophic factor required for endothelial cell survival and is abundantly expressed in the lung. METHODS AND RESULTS: We report that VEGF blockade decreases lung VEGF and VEGF receptor 2 (VEGFR-2) expression in newborn rats and impairs alveolar development, leading to alveolar simplification and loss of lung capillaries, mimicking BPD. In hyperoxia-induced BPD in newborn rats, air space enlargement and loss of lung capillaries are associated with decreased lung VEGF and VEGFR-2 expression. Postnatal intratracheal adenovirus-mediated VEGF gene therapy improves survival, promotes lung capillary formation, and preserves alveolar development in this model of irreversible lung injury. Combined VEGF and angiopoietin-1 gene transfer matures the new vasculature, reducing the vascular leakage seen in VEGF-induced capillaries. CONCLUSIONS: These findings underscore the importance of the vasculature in what is traditionally thought of as an airway disease and open new therapeutic avenues for lung diseases characterized by irreversible loss of alveoli through the modulation of angiogenic growth factors.

Adenoviridae↗

PubMed2005

Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by genetic and acquired abnormalities that suppress apoptosis and enhance cell proliferation in the vascular wall, including downregulation of the bone morphogenetic protein axis and voltage-gated K+ (Kv) channels. Survivin is an "inhibitor of apoptosis" protein, previously thought to be expressed primarily in cancer cells. We found that survivin was expressed in the pulmonary arteries (PAs) of 6 patients with PAH and rats with monocrotaline-induced PAH, but not in the PAs of 3 patients and rats without PAH. Gene therapy with inhalation of an adenovirus carrying a phosphorylation-deficient survivin mutant with dominant-negative properties reversed established monocrotaline-induced PAH and prolonged survival by 25%. The survivin mutant lowered pulmonary vascular resistance, RV hypertrophy, and PA medial hypertrophy. Both in vitro and in vivo, inhibition of survivin induced PA smooth muscle cell apoptosis, decreased proliferation, depolarized mitochondria, caused efflux of cytochrome c in the cytoplasm and translocation of apoptosis-inducing factor into the nucleus, and increased Kv channel current; the opposite effects were observed with gene transfer of WT survivin, both in vivo and in vitro. Inhibition of the inappropriate expression of survivin that accompanies human and experimental PAH is a novel therapeutic strategy that acts by inducing vascular mitochondria-dependent apoptosis.

Adenoviridae↗

PubMed2004

Oxygen-sensitive Kv channel gene transfer confers oxygen responsiveness to preterm rabbit and remodeled human ductus arteriosus: implications for infants with patent ductus arteriosus.

BACKGROUND: Oxygen (O2)-sensitive K+ channels mediate acute O2 sensing in many tissues. At birth, initial functional closure of the ductus arteriosus (DA) results from O2-induced vasoconstriction. This mechanism often fails in premature infants, resulting in persistent DA, a common form of congenital heart disease. We hypothesized that the basis for impaired O2 constriction in preterm DA is reduced expression and function of O2-sensitive, voltage-gated (Kv) channels. METHODS AND RESULTS: Preterm rabbit DA rings have reduced O2 constriction (even after inhibition of prostaglandin and nitric oxide synthases), and preterm DA smooth muscle cells (DASMCs) display reduced O2-sensitive K+ current. This is associated with decreased mRNA and protein expression of certain O2-sensitive Kv channels (Kv1.5 and Kv2.1) but equivalent expression of the L-type calcium channel. Transmural Kv1.5 or Kv2.1 gene transfer "rescues" the developmental deficiency, conferring O2 responsiveness to preterm rabbit DAs. Targeted SMC Kv1.5 gene transfer also enhances O2 constriction in human DAs. CONCLUSIONS: These data demonstrate a central role for developmentally regulated DASMC O2-sensitive Kv channels in the functional closure of the DA. Modulation of Kv channels may have therapeutic potential in diseases associated with impaired O2 responsiveness, including persistent DA.

4-Aminopyridine↗

PubMed2004

Preferential expression and function of voltage-gated, O2-sensitive K+ channels in resistance pulmonary arteries explains regional heterogeneity in hypoxic pulmonary vasoconstriction: ionic diversity in smooth muscle cells.

Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (E(M)), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (I(K)) and a more hyperpolarized E(M) and were uniquely O2- and correolide-sensitive. The O2-sensitive current (active at -65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited I(K) 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide's effects. The hypoxia-sensitive, correolide-insensitive I(K) (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2- and correolide-sensitive I(K) like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.

4-Aminopyridine↗

PubMed2003

In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats.

BACKGROUND: Alveolar hypoxia acutely elicits pulmonary vasoconstriction (HPV). Chronic hypoxia (CH), despite attenuating HPV, causes pulmonary hypertension (CH-PHT). HPV results, in part, from inhibition of O2-sensitive, voltage-gated potassium channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). CH decreases Kv channel current/expression and depolarizes and causes Ca2+ overload in PASMCs. We hypothesize that Kv gene transfer would normalize the pulmonary circulation (restore HPV and reduce CH-PHT), despite ongoing hypoxia. METHODS AND RESULTS: Adult male Sprague-Dawley rats were exposed to normoxia or CH for 3 to 4 weeks and then nebulized orotracheally with saline or adenovirus (Ad5) carrying genes for the reporter, green fluorescent protein reporter+/-human Kv1.5 (cloned from normal PA). HPV was assessed in isolated lungs. Hemodynamics, including Fick and thermodilution cardiac output, were measured in vivo 3 and 14 days after gene therapy by use of micromanometer-tipped catheters. Transgene expression, measured by quantitative RT-PCR, was confined to the lung, persisted for 2 to 3 weeks, and did not alter endogenous Kv1.5 levels. Ad5-Kv1.5 caused no mortality or morbidity, except for sporadic, mild elevation of liver transaminases. Ad5-Kv1.5 restored the O2-sensitive K+ current of PASMCs, normalized HPV, and reduced pulmonary vascular resistance. Pulmonary vascular resistance decreased at day 2 because of increased cardiac output, and remained reduced at day 14, at which time there was concomitant regression of right ventricular hypertrophy and PA medial hypertrophy. CONCLUSIONS: Kv1.5 is an important O2-sensitive channel and potential therapeutic target in PHT. Kv1.5 gene therapy restores HPV and improves PHT. This is, to the best of our knowledge, the first example of K+ channel gene therapy for a vascular disease.

Adenoviridae↗

PubMed2002

O2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor.

Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O2 alters the function of an O2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H2O2), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K+ channels, Kv). O2 constriction was evaluated in 26 human DAs (12 female, aged 9+/-2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O2 cause similar constriction and K+ current inhibition (I(K)). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O2 and 4-AP constriction in DA rings and reduced O2- and 4-AP-sensitive I(K) in DASMCs. Remodeled DAMSCs are depolarized and express less O2-sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BK(Ca)). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O2. In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing I(K) and reversing constriction to O2, but not phenylephrine. O2 increases, whereas hypoxia and ETC inhibitors decrease H2O2 production by altering mitochondrial membrane potential (DeltaPsim). H2O2, like O2, inhibits I(K) and depolarizes DASMCs. We conclude that O2 controls human DA tone by modulating the function of the mitochondrial ETC thereby varying DeltaPsim and the production of H2O2, which regulates DASMC Kv channel activity and DA tone.

4-Aminopyridine↗

PubMed2002

Diversity in mitochondrial function explains differences in vascular oxygen sensing.

Renal arteries (RAs) dilate in response to hypoxia, whereas the pulmonary arteries (PAs) constrict. In the PA, O2 tension is detected by an unidentified redox sensor, which controls K+ channel function and thus smooth muscle cell (SMC) membrane potential and cytosolic calcium. Mitochondria are important regulators of cellular redox status and are candidate vascular O2 sensors. Mitochondria-derived activated oxygen species (AOS), like H2O2, can diffuse to the cytoplasm and cause vasodilatation by activating sarcolemmal K+ channels. We hypothesize that mitochondrial diversity between vascular beds explains the opposing responses to hypoxia in PAs versus RAs. The effects of hypoxia and proximal electron transport chain (pETC) inhibitors (rotenone and antimycin A) were compared in rat isolated arteries, vascular SMCs, and perfused organs. Hypoxia and pETC inhibitors decrease production of AOS and outward K+ current and constrict PAs while increasing AOS production and outward K+ current and dilating RAs. At baseline, lung mitochondria have lower respiratory rates and higher rates of AOS and H2O2 production. Similarly, production of AOS and H2O2 is greater in PA versus RA rings. SMC mitochondrial membrane potential is more depolarized in PAs versus RAs. These differences relate in part to the lower expression of proximal ETC components and greater expression of mitochondrial manganese superoxide dismutase in PAs versus RAs. Differential regulation of a tonically produced, mitochondria-derived, vasodilating factor, possibly H2O2, can explain the opposing effects of hypoxia on the PAs versus RAs. We conclude that the PA and RA have different mitochondria.

Animals↗