Cost-effectiveness of insulin glargine versus sitagliptin in insulin-naïve patients with type 2 diabetes mellitus.

PURPOSE: In the EASIE (Evaluation of Insulin Glargine Versus Sitagliptin in Insulin-Naïve Patients) trial, insulin glargine found a significant reduction in glycosylated hemoglobin compared with sitagliptin in patients with type 2 diabetes who are inadequately controlled with metformin. The objective of this study was to assess the cost-effectiveness of insulin glargine compared with sitagliptin in type 2 diabetes patients, from the perspective of the publicly funded Canadian health care system. METHODS: The IMS CORE Diabetes Model, a standard Markov structure and Monte Carlo simulation model, was used. The model used a lifetime horizon to capture the long-term complications associated with type 2 diabetes. The efficacy of insulin glargine and sitagliptin in terms of glycosylated hemoglobin reduction and corresponding rates of hypoglycemia were obtained from the EASIE trial. Health utility and cost data were obtained from recently published Canadian publications. Univariate and probabilistic sensitivity analyses were conducted. FINDINGS: In the lifetime base-case analysis, treatment with insulin glargine resulted in cost savings of $1434 CAD in 2012 and a gain of 0.08 quality-adjusted life years per patient. A probabilistic sensitivity analysis found the robustness of the base-case analysis, with 88% probability of insulin glargine being dominant (ie, cost savings and more quality-adjusted life years). IMPLICATIONS: Insulin glargine is a clinically superior and cost-effective alternative to sitagliptin in patients with type 2 diabetes who are inadequately controlled with metformin.

Cost-effectiveness of ticagrelor versus clopidogrel in patients with acute coronary syndromes in Canada.

BACKGROUND: Ticagrelor demonstrated a significant reduction in major cardiac events in patients with acute coronary syndrome (ACS) compared with clopidogrel in the Platelet Inhibition and Patient Outcomes (PLATO) trial. The objective of this study was to assess the cost-effectiveness of ticagrelor compared with clopidogrel in ACS patients from the perspective of the Canadian publicly funded health care system. METHODS: A two-part model was developed consisting of a 1-year decision tree and a lifetime Markov model. Within the decision tree, patients remained event-free, experienced a nonfatal myocardial infarction, a nonfatal stroke, or death due to vascular or nonvascular related causes based on data from the PLATO trial. The lifetime Markov model followed these patients and allowed for subsequent myocardial infarction, stroke, and death. Patient utility and resource use were derived from the PLATO trial. Transition probabilities and specific Canadian unit costs were derived from published sources. Univariate and probabilistic sensitivity analyses were conducted. RESULTS: In the base case lifetime analysis, treatment with ticagrelor resulted in more years of life per person (0.097), more quality-adjusted life years per person (QALYs, 0.084), and an incremental cost per QALY gained of $9,745 (Canadian$), assuming a generic cost for clopidogrel. A probabilistic sensitivity analysis demonstrated the robustness of the base case analysis, with a 93% probability of being below $20,000 per QALY gained and a 99% probability of being below $30,000 per QALY gained. CONCLUSION: Ticagrelor is a clinically superior and cost-effective option for the prevention of thrombotic events among ACS patients in Canada.

Chronic nicotine induces hypoxia inducible factor-2α in perinatal rat adrenal chromaffin cells: role in transcriptional upregulation of KATP channel subunit Kir6.2.

Fetal nicotine exposure causes impaired adrenal catecholamine secretion and increased neonatal mortality during acute hypoxic challenges. Both effects are attributable to upregulation of ATP-sensitive K(+) channels (K(ATP) channels) and can be rescued by pretreatment with the blocker, glibenclamide. Although use of in vitro models of primary and immortalized, fetal-derived rat adrenomedullary chromaffin cells (i.e., MAH cells) demonstrated the involvement of α7 nicotinic ACh receptor (nAChR) stimulation and the transcription factor, HIF-2α, the latter's role was unclear. Using Western blots, we show that chronic nicotine causes a progressive, time-dependent induction of HIF-2α in MAH cells that parallels the upregulation of K(ATP) channel subunit, Kir6.2. Moreover, a common HIF target, VEGF mRNA, was also upregulated after chronic nicotine. All the above effects were prevented during co-incubation with α-bungarotoxin (100 nM), a specific α7 nAChR blocker, and were absent in HIF-2α-deficient MAH cells. Chromatin immunoprecipitation (ChIP) assays demonstrated binding of HIF-2α to a putative hypoxia response element in Kir6.2 gene promoter. Specificity of this signaling pathway was validated in adrenal glands from pups born to dams exposed to nicotine throughout gestation; the upregulation of both HIF-2α and Kir6.2 was confined to medullary, but not cortical, tissue. This study has uncovered a signaling pathway whereby a nonhypoxic stimulus (nicotine) promotes HIF-2α-mediated transcriptional upregulation of a novel target, Kir6.2 subunit. The data suggest that the HIF pathway may be involved in K(ATP) channel-mediated neuroprotection during brain ischemia, and in the effects of chronic nicotine on ubiquitous brain α7 nAChR.

Chronic hypoxia upregulates adenosine 2a receptor expression in chromaffin cells via hypoxia inducible factor-2α: role in modulating secretion.

Catecholamine (CAT) release from chromaffin tissue plays an essential role in the fetus which develops in a low O2 environment (hypoxia). To address molecular mechanisms regulating CAT secretion in low O2, we exposed a fetal chromaffin-derived cell line (MAH cells) to chronic hypoxia (CHox; 2% O2, 24h) and assessed gene expression using microarrays, quantitative RT-PCR, and western blot. CHox caused a dramatic ∼12× upregulation of adenosine A2a receptor (A2aR) mRNA, an effect critically dependent upon hypoxia-inducible factor (HIF)-2α which bound the promoter of the A2aR gene. In amperometric studies, acute hypoxia and high K⁺ (30 mM) evoked quantal CAT secretion that was enhanced after CHox, and further potentiated during simultaneous A2aR activation by adenosine. A2aR activation also enhanced stimulus-induced rise in intracellular Ca²âº in control, but not HIF-2α-deficient, MAH cells. Thus, A2aR, adenosine, and HIF-2α are key contributors to the potentiation of CAT secretion in developing chromaffin cells during chronic hypoxia.

Divergent roles of reactive oxygen species in the responses of perinatal adrenal chromaffin cells to hypoxic challenges.

The fetus and neonate experience variable patterns of low P(O)2(hypoxia) ranging from acute, sustained, and intermittent. Adaptation to hypoxia involves activation of key transcription factors, known as hypoxia-inducible factors (e.g. HIF-1α, HIF-2α), which regulate a number of genes in different cell types. This review focuses on the signaling pathways that mediate proper physiological responses of perinatal adrenomedullary chromaffin cells (AMC) to varying patterns of hypoxic challenges, and particularly on the controversial role of reactive oxygen species (ROS). At birth, acute hypoxia (seconds to minutes) directly stimulates catecholamine release from AMC via K+ channel inhibition, mediated by a decrease in mitochondrial-derived ROS. By contrast, exposure to chronic sustained hypoxia (CSH) induces HIF-2α in a fetal-derived chromaffin cell line independently of changes in ROS. Exposure to chronic intermittent hypoxia (CIH) activates antioxidant responses via the regulator Nrf-2, in association with an increase in ROS and the induction of HIF-1α. We propose that the physiological responses of perinatal AMC to hypoxia and the ensuing directional changes in ROS are dependent on the pattern and duration of the hypoxic exposure.

Hypoxia inducible factor (HIF)-2 alpha is required for the development of the catecholaminergic phenotype of sympathoadrenal cells.

The basic helix-loop-helix transcription factor, hypoxia inducible factor (HIF)-2alpha has been implicated in the development of the catecholaminergic phenotype in cells of the sympathoadrenal (SA) lineage; however, the underlying mechanisms and HIF-2alpha targets remain unclear. Using an immortalized rat adrenomedullary chromaffin cell line (MAH cells) derived from a fetal SA progenitor, we examined the role of HIF-2alpha in catecholamine biosynthesis. Chronic hypoxia (2% O(2), 24 h) induced HIF-2alpha in MAH cells but expression of the rate-limiting enzyme, tyrosine hydroxylase (TH) and catecholamine levels were unaltered. Interestingly, HIF-2alpha depleted MAH cells showed dramatically lower (5-12 times) levels of dopamine and noradrenaline compared with wild-type and scrambled controls, even in normoxia (21% O(2)). This was correlated with a marked reduction in the expression of DOPA decarboxylase (DDC) and dopamine beta hydroxylase (DbetaH) but not TH. Chromatin immunoprecipitation assays revealed that HIF-2alpha was bound to the DDC gene promoter which contains two putative hypoxia response elements. These data suggest that a basal level of HIF-2alpha function is required for the normal developmental expression of DDC and DbetaH in SA progenitor cells, and that loss of this function leads to impaired catecholamine biosynthesis.

Glucosensing in an immortalized adrenomedullary chromaffin cell line: role of ATP-sensitive K+ channels.

Using an immortalized adrenal chromaffin cell line (MAH cells), we investigated the cellular mechanisms underlying sensitivity to glucose-free solution (aglycemia) using ratiometric Ca2+ imaging and whole-cell recording. Though few cells (< 15%) responded to aglycemia with an increase in intracellular-free Ca2+ concentration ([Ca2+]i), in most cells (approximately 75%), aglycemia caused > 50% suppression of the Delta[Ca2+]i induced by the depolarizing stimulus, high (10 mM) K+. Moreover, in normal K+, the average aglycemia-induced rise in Cai2+ as well as the proportion of aglycemia-responsive cells increased in the presence of the K(ATP) channel blocker, glibenclamide. During membrane potential (Vm) measurements, aglycemia induced either hyperpolarization (6/20), depolarization (4/20) or no change in Vm. RT-PCR and Western blotting confirmed the presence of K(ATP) channel subunits Kir6.2 and SUR1 in MAH cells. These findings suggest a dual inhibitory and excitatory action of aglycemia in MAH cells, where activation of K(ATP) channels effectively inhibits or blunts the Delta[Ca2+]i due to the excitatory effect. Thus, this cell line appears as an attractive model for studying the molecular mechanisms of glucosensing.

Induction of HIF-2alpha is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line.

During low O2 (hypoxia), hypoxia-inducible factor (HIF)-alpha is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2alpha expression and oxygen consumption in an O2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2-8% O2) caused a concentration- and time-dependent increase in HIF-2alpha induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA (rho0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 microM), myxothiazol (1 microM), antimycin A (1 microg/ml), and cyanide (1 mM), blocked HIF-2alpha induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O2 concentration such that at moderate-to-severe hypoxia (6% O2), HIF-2alpha induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2alpha induction. These data suggest that in this O2-sensitive cell line, mitochondrial O2 consumption, rather than changes in ROS, regulates HIF-2alpha during hypoxia.

An EGR2/CITED1 transcription factor complex and the 14-3-3sigma tumor suppressor are involved in regulating ErbB2 expression in a transgenic-mouse model of human breast cancer.

Amplification and elevated expression of the ErbB2 receptor tyrosine kinase occurs in 20% of human breast cancers and is associated with a poor prognosis. We have previously demonstrated that mammary tissue-specific expression of activated ErbB2 under the control of its endogenous promoter results in mammary tumor formation. Tumor development was associated with amplification and overexpression of ErbB2 at both the transcript and protein levels. Here we demonstrate that the EGR2/Krox20 transcription factor and its coactivator CITED1 are coordinately upregulated during ErbB2 tumor induction. We have identified an EGR2 binding site in the erbB2 promoter and demonstrated by chromatin immunoprecipitation assays that EGR2 and CITED1 associate specifically with this region of the promoter. EGR2 and CITED1 were shown to associate, and expression from an erbB2 promoter-reporter construct was stimulated by EGR2 and was further enhanced by CITED1 coexpression. Furthermore, expression of the 14-3-3sigma tumor suppressor led to downregulation of ErbB2 protein levels and relocalization of EGR2 from the nucleus to the cytoplasm. Taken together, these observations suggest that, in addition to an increased gene copy number and upregulation of EGR2 and CITED1, an elevated erbB2 transcript level involves the loss of 14-3-3sigma, which sequesters a key transcriptional regulator of the erbB2 promoter.

Distinct roles for hindbrain and paraxial mesoderm in the induction and patterning of the inner ear revealed by a study of vitamin-A-deficient quail.

The hindbrain and cranial paraxial mesoderm have been implicated in the induction and patterning of the inner ear, but the precise role of the two tissues in these processes is still not clear. We have addressed these questions using the vitamin-A-deficient (VAD) quail model, in which VAD embryos lack the posterior half of the hindbrain that normally lies next to the inner ear. Using a battery of molecular markers, we show that the anlagen of the inner ear, the otic placode, is induced in VAD embryos in the absence of the posterior hindbrain. By performing grafting and ablation experiments in chick embryos, we also show that cranial paraxial mesoderm which normally lies beneath the presumptive otic placode is necessary for otic placode induction and that paraxial mesoderm from other locations cannot induce the otic placode. Two members of the fibroblast growth factor family, FGF3 and FGF19, continue to be expressed in this mesodermal population in VAD embryos, and these may be responsible for otic placode induction in the absence of the posterior hindbrain. Although the posterior hindbrain is not required for otic placode induction in VAD embryos, the subsequent patterning of the inner ear is severely disrupted. Several regional markers of the inner ear, such as Pax2, EphA4, SOHo1 and Wnt3a, are incorrectly expressed in VAD otocysts, and the sensory patches and vestibulo-acoustic ganglia are either greatly reduced or absent. Exogenous application of retinoic acid prior to 30 h of development is able rescue the VAD phenotype. By performing such rescue experiments before and after 30 h of development, we show that the inner ear defects of VAD embryos correlate with the absence of the posterior hindbrain. These results show that induction and patterning of the inner ear are governed by separate developmental processes that can be experimentally uncoupled from each other.

Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain.

The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.

Dlx gene expression during chick inner ear development.

Members of the Dlx gene family play essential roles in the development of the zebrafish and mouse inner ear, but little is known regarding Dlx genes and avian inner ear development. We have examined the inner ear expression patterns of Dlx1, Dlx2, Dlx3, Dlx5, and Dlx6 during the first 7 days of chicken embryonic development. Dlx1 and Dlx2 expression was seen only in nonneuronal cells of the cochleovestibular ganglion and nerves from stage 21 to stage 32. Dlx3 marks the otic placode beginning at stage 9 and becomes limited to epithelium adjacent to the hindbrain as invagination of the placode begins. Dlx3 expression then resolves to the dorsal otocyst and gradually becomes limited to the endolymphatic sac by stage 30. Dlx5 and Dlx6 expression in the developing inner ear is first seen at stages 12 and 13, respectively, in the rim of the otic pit, before spreading throughout the dorsal otocyst. As morphogenesis proceeds, Dlx5 and Dlx6 expression is seen throughout the forming semicircular canals and endolymphatic structures. During later stages, both genes are seen to mark the distal surface of the forming canals and display expression complementary to that of BMP4 in the vestibular sensory regions. Dlx5 expression is also seen in the lagena macula and the cochlear and vestibular nerves by stage 30. These findings suggest important roles for Dlx genes in the vestibular and neural development of the avian inner ear.

Amyloid beta peptides mediate physiological remodelling of the acute O2 sensitivity of adrenomedullary chromaffin cells following chronic hypoxia.

OBJECTIVE: The non-neurogenic response of the neonatal adrenal medulla is vital in cardiovascular and respiratory development and to the survival of newborns exposed to hypoxic stress. Here, we examined the acute hypoxic response of immortalised rat adrenomedullary chromaffin cells following exposure to chronic hypoxia (CH; 6% O(2) for 24 h). METHODS: Ca(2+) and K(+) channel currents were recorded using by whole-cell patch-clamp. RESULTS: Following incubation in CH, the acute O(2) sensitivity of K(+) current in immortalised adrenomedullary chromaffin (MAH) cells was enhanced due to a selective increase in the density of an O(2)-sensitive Ca(2+)-dependent K(+) current, secondary to ROS-mediated augmentation of voltage-gated Ca(2+) currents. The effect of CH on Ca(2+) currents was not additive to exogenous Abeta(1-40) and was blocked by the gamma-secretase inhibitors gamma-X and gamma-VI, demonstrating a role for amyloid beta peptide (AbetaP) production. Ca(2+) current enhancement was abolished in the presence of the transcription inhibitor actinomycin D but unaffected by the vacuolar H(+) ATPase inhibitor bafilomycin A1. CONCLUSION: AbetaP production and transcriptional regulation during CH regulated the properties of a peripheral chemosensory cell, defining a role for these enigmatic peptides in the signalling pathway of a physiological response to CH in the developing cardiovascular system.

Acute and chronic hypoxic regulation of recombinant hNa(v)1.5 alpha subunits.

Acute and chronic hypoxic regulation of ion channels is involved in both cell physiology and pathology. Voltage-dependent Na(+) channels play a dominant role in the upstroke of the action potential in excitable cells, while non-inactivating (persistent or sustained) Na(+) currents contribute to action potential shape and duration. In cardiac myocytes, hypoxic augmentation of persistent Na(+) currents has been proposed to underlie cardiac arrhythmias via prolonging action potential duration. Here, we demonstrate that acute hypoxia (P(O2), 20mm Hg) augmented persistent Na(+) currents in HEK293 cells stably expressing human Na(v)1.5 alpha subunits. Hypoxia also inhibited peak Na(+) currents in a voltage-dependent manner, and the kinetics of activation and inactivation of Na(+) currents were significantly slowed during hypoxia. We further demonstrate that exposure to chronic hypoxia (6% O(2) for 24h) augmented peak Na(+) channel current, which given the exogenous promoter driving expression of the channel occurs most probably via a post-transcriptional mechanism. These effects of acute and chronic hypoxia likely play an arrhythmogenic role during both short- and long-term hypoxic/ischaemic episodes. The HEK293 expression system provides a useful paradigm in which to examine the mechanisms of O(2) sensing by the Na(+) channel.

H2O2 regulates recombinant Ca2+ channel alpha1C subunits but does not mediate their sensitivity to acute hypoxia.

Acute hypoxic inhibition of the pore-forming alpha(1C) subunit of the L-type Ca(2+) channel mediates hypoxic arterial vasodilatation, a physiological response which matches tissue O(2) demand and supply in the systemic vasculature. In numerous O(2)-sensing cell types, reactive O(2) species (ROS) have been proposed as mediators linking lowered O(2) levels with the appropriate cellular response. In this study, we examined the roles of H(2)O(2) and NADPH oxidase as mediators of hypoxic inhibition of recombinant alpha(1C) subunits. Human cardiac L-type Ca(2+) channel alpha(1C) subunits were stably expressed in HEK 293 cells. Ca(2+) currents were recorded using the whole-cell configuration of the patch-clamp technique. Bath application of 100microM H(2)O(2) significantly enhanced depolarisation-evoked Ca(2+) currents in a voltage-dependent manner, while dialysis with 1000Uml(-1) catalase reduced these currents. In the presence of catalase, hypoxic inhibition of Ca(2+) currents was not significantly different compared to non-dialysed controls. The NADPH oxidase inhibitors diphenylene iodonium (10microM) and phenylarsine oxide (5microM) were without effect on either basal Ca(2+) currents or responses to hypoxia. Thus, endogenous production of H(2)O(2) regulates the alpha(1C) subunit. However, neither suppression of H(2)O(2) levels nor inhibition of NADPH oxidase is involved in O(2)-dependent regulation of the Ca(2+) channel.