Diabetes and Vascular Disease Research http://dvr.sagepub.com/ Glucose attenuates hypoxia-induced changes in endothelial cell growth by inhibiting HIF-1α expression Wei Gao, Gail Ferguson, Paul Connell, Tony Walshe, Colm O'Brien, Eileen M Redmond and Paul A Cahill Diabetes and Vascular Disease Research 2014 11: 270 originally published online 22 May 2014 DOI: 10.1177/1479164114533356 The online version of this article can be found at: http://dvr.sagepub.com/content/11/4/270
Published by: http://www.sagepublications.com
Additional services and information for Diabetes and Vascular Disease Research can be found at: Email Alerts: http://dvr.sagepub.com/cgi/alerts Subscriptions: http://dvr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Jun 16, 2014 OnlineFirst Version of Record - May 22, 2014 What is This?
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
533356 research-article2014
DVR0010.1177/1479164114533356Diabetes & Vascular Disease ResearchGao et al.
Original Article
Glucose attenuates hypoxia-induced changes in endothelial cell growth by inhibiting HIF-1α expression
Diabetes & Vascular Disease Research 2014, Vol. 11(4) 270–280 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1479164114533356 dvr.sagepub.com
Wei Gao1, Gail Ferguson1, Paul Connell1,2, Tony Walshe1, Colm O’Brien2, Eileen M Redmond3 and Paul A Cahill1
Abstract Hyperglycaemia and hypoxia play essential pathophysiological roles in diabetes. We determined whether hyperglycaemia influences endothelial cell growth under hypoxic conditions in vitro. Using a Ruskinn Invivo2 400 Hypoxia Workstation, bovine aortic endothelial cells (BAEC) were exposed to high glucose concentrations (25 mM glucose) under normoxic or hypoxic conditions before cell growth (balance of proliferation and apoptosis) was assessed by fluorescence-activated cell sorting (FACS) analysis, proliferating cell nuclear antigen (pCNA), Bcl-xL and caspase-3 protein expression and activity. Hypoxia increased hypoxia response element (HRE) transactivation and induced hypoxia-inducible factor-1α (HIF-1α) expression when compared to normoxic controls concomitant with a significant decrease in cell growth. High glucose (25 mM) concentrations attenuated HRE transactivation and HIF-1α protein expression while concurrently reducing hypoxia-induced changes in BAEC growth. Knockdown of HIF-1α expression significantly decreased hypoxiainduced changes in growth and attenuated the modulatory effects of glucose. These results provide evidence that hypoxia-induced control of BAEC growth can be altered by the presence of glucose via inhibition of HIF-1α expression and activation. Keywords Endothelial, hypoxia, hyperglycaemia, HIF-1α, apoptosis, diabetes
Introduction Diabetes mellitus currently affects more than 170 million individuals worldwide and is expected to afflict another 200 million individuals in the next 30 years.1 The majority of diabetic morbidity relates to the incidence of cardiovascular disease where the diabetic patient cohort suffers from accelerated and severe atherosclerosis that can lead to heart attack, stroke and peripheral vascular disease.2 While diabetes is an established risk factor for vascular disease such as atherosclerosis, the mechanism by which diabetes accelerates the disease remains unknown. Glucose fluctuations characteristic of both Type I and Type II diabetes have been implicated in diabetic atherosclerosis as tight glycaemic control has been shown to significantly reduce the risk of myocardial infarction and stroke in people with diabetes.3,4 Hypoxia is a reduction in oxygen delivery below tissue demand and can be either acute or chronic. It is centrally regulated by hypoxia-inducible factor (HIF), a heterodimer composed of α and β subunits that belong to the basic helix–loop–helix (bHLH) Per, Arnt, Sim (PAS) family of transcription factors.5,6 While severe chronic hypoxia can
cause cell death, more modest hypoxia can protect against subsequent cell damage.7 The intensity and sustainability of HIF-1α activation are considered one of the major determinants of whether responses are pathological or beneficial. Several important processes are characterized by hypoxia, including ischaemia–reperfusion, tumour growth and progression, inflammation, myocardial ischaemia and a number of ocular pathologies.7,8
1Vascular
Biology and Therapeutics Laboratory, School of Biotechnology, Faculty of Science and Health, Dublin City University, Dublin, Ireland 2Mater Misericordiae Hospital, Institute of Ophthalmology, The Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland 3Department of Surgery, University of Rochester, Rochester, NY, USA Corresponding author: Paul A Cahill, Vascular Biology and Therapeutics Laboratory, School of Biotechnology, Faculty of Science and Health, Dublin City University, Dublin 9, Ireland. Email: [email protected]
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
271
Gao et al. There have been numerous studies demonstrating reduced levels of HIF-α from patients with diabetes and animal models of diabetes in conjunction with mammalian cells cultured in high glucose medium.9–11 Hyperglycaemia appears to compromise HIF-α protein levels and transactivation function, yet the detailed molecular mechanisms underlying impairment of HIF-α by high glucose remain poorly understood. It is known that HIF-1α regulates the expression of enzymes involved in the process of glycolysis and of GLUT1 and GLUT3 which mediate cellular glucose uptake.12 On the other hand, glucose, glucose uptake and glycolysis influence the stability and activation of HIF-1α.13,14 In this study, we examined the effects of high glucose levels on endothelial cell growth (the balance of proliferation and apoptosis) under normoxic and hypoxic conditions while investigating the putative role of HIF-1α in this response. Our data provide evidence that hypoxia-induced changes in endothelial cell growth can be attenuated by glucose through modulation of HIF-1α.
Methods Materials All materials were of the highest purity and were purchased from Sigma–Aldrich unless otherwise stated. Antibodies were purchased from the vendors for proliferating cell nuclear antigen (pCNA) (1:80; Cayman Chemical Co., Ann Arbor, MI, USA), caspase-3 (1:1000; Cell Signaling Technology, MSC, Ireland), Bcl-xL (1:200; BD Biosciences Europe, UK), HIF-1α (1:1000; BD Biosciences) or BNIP3L [1:500; Axxora (UK) Ltd].
Cell culture Bovine aortic endothelial cells (BAEC) were purchased from the Coriell Cell Repositories (Cat AG07680) and grown in normal glucose (control: 5.5 mM) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. Cell lines were maintained in a humidified atmosphere of 5% CO2/95% air (normoxic conditions) and routinely used between passages 9 and 14.
Hypoxic/high-glucose studies Cells were quiesced for 48 h in control DMEM supplemented with 0.5% FBS before cells were exposed to normoxic (5% CO2, 95% air) or chronic hypoxic (2% O2, 5% CO2, 93% N2) conditions in DMEM supplemented with 10% FBS in the presence of normal glucose (C: control: 5.5 mM) or high glucose (G: glucose: 25 mM) or a mannitol control (M: mannitol 25 mM) for 5 days. Media were replaced daily using the Ruskinn Invivo2 400 Hypoxia Workstation as previously described.13
Proliferation and apoptosis assays Effects on cell proliferation and apoptosis were determined using the Vybrant® CFDA SE Cell Proliferation Kit and the Vybrant Apoptosis Assay Kit #2 (Molecular Probes®, Invitrogen, Ireland) followed by fluorescence-activated cell sorting (FACS) analysis using a FACSCAN flow cytometer (Becton, Dickinson and Company, Oxford UK) as previously described.13,15 Apoptosis was also determined by measuring caspase-3 activity and expression using an assay based on the cleavage of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) and an anti-caspase-3 antibody, respectively, as previously described by us.13
Western blotting Soluble extracts (15–40 µg) were fractionated by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) on 6%–12% (w/v) polyacrylamide resolving gels depending on protein size to be detected as previously described.13 Scanning densitometry was performed with image analysis software [one-dimensional (1D); Eastman-Kodak, Rochester, NY, USA]. Minor differences in protein loading and transfer were normalized using a Ponceau S stain as previously described16 and validated as before.17
Semi-quantitative reverse transcriptionpolymerase chain reaction Total RNA was isolated from cells using TRIzol™ (Invitrogen) according to the manufacturer’s specifications and was reverse transcribed to complementary DNA (cDNA) using iScript as per the manufacturer’s specifications (Alpha Technologies, UK) as previously described.13
Transient transfections BAEC were plated in six-well plates and incubated for 3 h in 1 mL/well DMEM containing 2 µg of cDNA or 100 µg/ mL scrambled or small interfering RNA (siRNA)-specific duplexes and 4 µL of Lipofectamine™ (Invitrogen™, BioSciences Ltd, Ireland) according to the manufacturer’s instructions. The siRNA duplexes for HIF-1α corresponded to positions 1254–1274 in the bovine HIF-1α sequence (accession number: NM_174339) and were acquired from M W G Biotech (UK). Scrambled siRNA duplexes were used as controls. The hypoxia response element (HRE)-luciferase reporter plasmid was a kind gift from Dr Cormac Taylor, Conway Institute of Biomolecular & Biomedical Research, Ireland. DNA transfection efficiency was confirmed and normalized to β-galactosidase activity following co-transfection with CMV-LacZ, a plasmid-encoding β-galactosidase activity as previously described.13
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
272
Diabetes & Vascular Disease Research 11(4)
Statistical analysis All treatments were normalized to quiesced cells (serumdeprived control at day 0) and expressed as a fold increase or decrease compared to this value. The results are expressed as mean ± standard error of mean (SEM). Statistical significance was assessed by analysis of variance (ANOVA) with significance values of p ≤ 0.01 and p ≤ 0.05 for difference between groups.
Results BAEC were initially grown in control DMEM containing glucose (5.5 mM) before the cells were exposed to normoxic or hypoxic conditions for 5 days. HRE-luciferase activity was negligible under normoxic conditions. However, following exposure to hypoxia, HRE transactivation increased significantly, an effect that was significantly attenuated with glucose (25 mM) (Figure 1(a)). In parallel cultures, HIF-1α protein subunit levels were undetectable after 5 days under normoxic conditions (Figure 1(b)) but increased dramatically after 5 days under hypoxic conditions (Figure 1(b)). HIF-1α protein expression was significantly attenuated, without any significant change in HIF-1α messenger RNA (mRNA) levels, when cells were exposed to hypoxia in the presence of glucose (25 mM) (Figure 1(b) and (c)). The effect of hypoxia and hyperglycaemia on BAEC proliferation was initially examined by FACS analysis using a carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) fluorescent cell assay. Exposure of cells to 0.5% and 10% foetal calf serum (FCS) for 5 days confirmed that a shift to the left in the CFDA FACS scatter plot represented a significant increase in cell proliferation (data not shown). In response to hypoxia, there was a dramatic shift to the right representing a decrease in cell proliferation when compared to normoxia (Figure 2(a)). While glucose (25 mM) did not alter the proliferation state of the cells under normoxia (Figure 2(b)), it did result in a significant increase in cell proliferation following hypoxia (Figure 2(c)). Parallel studies confirmed that the number of cells significantly increased at day 5 when compared to C (t = 0). Moreover, under normoxic conditions, the number of cells after 5 days was similar following treatment with high glucose (G: 25 mM) when compared to control glucose (C: 5.5 mM) or mannitol (M: 25 mM) treatment (Figure 2(d)). In contrast, hypoxia resulted in a significant decrease in cell number, an effect that was attenuated following exposure to glucose (25 mM) (Figure 2(d)). Similarly, the level of pCNA expression, a cyclin marker of cell proliferation, was increased after 5 days under normoxia when compared to day 0 but was unaffected following treatment with glucose. However, hypoxia significantly decreased pCNA expression, an effect that was attenuated by the addition of glucose (Figure 2(e)).
Figure 1. Effect of hyperglycaemia and hypoxia on HIF-1α activity and expression: (a) hypoxia-induced HRE-luciferase transactivation under normoxic and hypoxic conditions (n = 3 ± SEM, #p ≤ 0.01 compared to normoxic control, *p ≤ 0.01 compared to hypoxia mannitol); (b) representative immunoblot and cumulative data of HIF-1α expression in BAEC under normoxic and hypoxic conditions (n = 3 ± SEM, *p ≤ 0.01 compared to mannitol) and (c) representative qRT-PCR of HIF-1α mRNA levels in BAEC under normoxic and hypoxic conditions [C, control (5.5 mM glucose), G (25 mM glucose) and M (25 mM mannitol)].
HIF-1α: hypoxia-inducible factor-1α; HRE: hypoxia response element; SEM: standard error of mean; BAEC: bovine aortic endothelial cells; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; mRNA: messenger RNA.
As growth is the balance of proliferation and apoptosis, we also examined the effect of hypoxia on BAEC apoptosis in the absence or presence of glucose. There was no significant change in apoptosis following treatment of
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
273
Gao et al.
Figure 2. Effect of hyperglycaemia on hypoxia-induced changes in BAEC proliferation: (a) representative scatter data obtained from FACS analysis of CFDA staining for BAEC proliferation at days 0 and 5 under normoxic and hypoxic conditions using the Vybrant® CFDA SE Cell Tracer Kit. Representative scatter data obtained from FACS analysis of BAEC proliferation under (b) normoxic and (c) hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol. (d) Cumulative cell count data of BAEC proliferation under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol and (e) Western blot analysis of concurrent cell lysates for pCNA in the presence or absence of glucose and mannitol (25 mM). Data are the mean ± SEM for three independent experiments performed in triplicate (#p ≤ 0.01 compared to day 0, *p ≤ 0.01 compared to normoxic control and ¶p ≤ 0.01 compared to hypoxia mannitol) [C (t = 0) = quiesced cells at time 0: prior to treatment; C = 10% FBS media; G = 10% FBS media + 25 mM glucose; M = 10% FBS media + 25 mM mannitol]. BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting; CFDA: carboxyfluorescein diacetate; pCNA: proliferating cell nuclear antigen; SEM: standard error of mean; FBS: foetal bovine serum.
cells with high glucose levels (25 mM) under normoxia (Figure 3(a)). In contrast, hypoxia promoted apoptosis, an effect that was reduced in the presence of glucose (Figure 3(a)). Further analysis of apoptosis by colorimetric measurement of caspase-3 activity and immunoblot detection of caspase-3 protein levels confirmed that the hypoxiainduced changes in caspase-3 activity and protein expression were attenuated in the presence of glucose (Figure 3(b) and (c)).
The effects of hypoxia on the stress activator response18 were also investigated. Hypoxia caused a reduction in the expression of the anti-apoptotic protein, Bcl-xL, when compared to normoxia (Figure 3(d)), without any change in Bcl-xL mRNA levels (data not shown). However, the decrease in Bcl-xL protein expression was attenuated by glucose (25 mM) (Figure 3(d)), again without any change in Bcl-xL mRNA levels (data not shown). Further analysis of the expression of the pro-apoptotic protein BNIP3L, a
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
274
Diabetes & Vascular Disease Research 11(4)
Figure 3. Effect of hyperglycaemia on hypoxia-induced changes in BAEC apoptosis: (a) representative data obtained from FACS analysis of BAEC apoptosis using the Vybrant® Apoptosis Assay Kit (inset: V = viable, EA = early apoptotic, LA = late apoptotic, N = necrotic). The values obtained for EA and LA cells were combined and plotted as a bar chart: (b) caspase-3 enzyme activity and (c) representative immunoblot of caspase-3 expression under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol, and (d) effect of hyperglycaemia and hypoxia on Bcl-xL expression in BAEC. Representative immunoblot and cumulative data of Bcl-xL and BNIP3L expression in BAEC under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and mannitol (n = 3, #p ≤ 0.01 compared to normoxic control, *p ≤ 0.01 compared to hypoxic mannitol). BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting.
natural suppressor of Bcl-xL activity, confirmed that hypoxia promoted BNIP3L protein expression (Figure 3(d)), a response that was significantly reduced by glucose (Figure 3(d)). The putative role of HIF-1α in regulating hypoxiainduced changes in BAEC growth (balance of proliferation and apoptosis) was examined in cells following HIF-1α knockdown. Initial quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis
confirmed HIF-1α knockdown (Figure 4(a)). BAEC proliferation remained unchanged in cells transfected with either HIF-1α or scrambled siRNA under normoxia when cells were treated with glucose (25 mM) (Figure 5(a)). In contrast, hypoxia inhibited BAEC proliferation in cells transfected with scrambled siRNA (Figure 5(b) vs 5(a)), an effect that was attenuated by glucose (Figure 5(b)). However, this recovery in BAEC proliferation by glucose (25 mM) was abolished following HIF-1α knockdown (Figure 5(b)). In
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
275
Gao et al.
transfected with scrambled siRNA (Figure 6(a)), an effect that was significantly reduced following HIF-1α knockdown (Figure 6(a)). Furthermore, the level of apoptosis was unchanged following treatment of cells with glucose (Figure 6(a)). In parallel cultures, caspase-3 activity was also unchanged following scrambled siRNA or HIF-1α knockdown under normoxia (Figure 6(b)) with or without glucose (Figure 6(b)). However, hypoxia increased caspase-3 activity in scrambled siRNA-transfected cells (Figure 6(b)), an effect that was significantly reduced following knockdown of HIF-1α (Figure 6(b)). Moreover, caspase-3 activity was not significantly further reduced following treatment of cells with glucose (Figure 6(b)).
Discussion
Figure 4. HIF-1α gene silencing inhibits hypoxia-induced HIF-1α: (a) HIF-1α mRNA levels in cells transfected with HIF1α siRNA compared to scrambled and NT controls [n = 3 ± SEM, *p ≤ 0.01 compared to scrambled (Scram) control] and (b) HIF-1α protein expression in cells transfected with HIF-1α siRNA compared to scrambled and NT controls [n = 3 ± SEM, *p ≤ 0.01 compared to scrambled (Scram) control].
HIF-1α: hypoxia-inducible factor-1α; siRNA: small interfering RNA; NT: non-transfected; SEM: standard error of mean; mRNA: messenger RNA.
parallel studies, hypoxia promoted a decrease in cell number in scrambled controls, an effect that was attenuated by glucose. However, when cells were transfected with siRNA against HIF-1α, there was no significant change in cell number under normoxia or hypoxia (Figure 5(c)). In addition, there was no significant difference in cell number following treatment with glucose (Figure 5(c)). HIF-1α knockdown resulted in a modest reduction in the baseline level of apoptosis under normoxia (Figure 6(a)). There was no significant effect of glucose (Figure 6(a)). In contrast, hypoxia promoted apoptosis in BAEC
The causative factors of diabetic retinopathy,19 neuropathy20 and arteriosclerosis6,21 include hyperglycaemia and hypoxia. This is primarily because blood glucose is strongly associated with morbidity after an acute hypoxic challenge, that is, acute myocardial infarction, suggesting a potential deleterious influence of hyperglycaemia on the tissue’s capacity to adapt to low oxygen tensions.22 As a result, there exists a close interrelationship between glucose and HIF-1α in several biological systems. HIF-1α regulates the expression of numerous enzymes involved in the process of glycolysis,12 whereas glucose uptake and glycolysis can significantly influence the stability and activation of HIF-1α.6,8,23 Hypoxia-induced HIF-1α activation is very well characterized for numerous cell types,24 including vascular endothelial cells.7 Glucose has also been shown to affect HIF-1α expression and activity in several cell types11,25 including vascular smooth muscle cells.13 We therefore assessed the effect of hyperglycaemia on HIF-1α expression and activity in endothelial cells. Several studies have demonstrated that altered glucose levels contribute to the endothelial cell dysfunction,26–28 which is a major initiating step in vascular disease.29 Healthy endothelial cells maintain vascular homeostasis through tight control of permeability, inflammation, vascular tone and injury repair responses.30 However, high glucose levels can promote increased endothelial cell permeability, allowing solutes to pass into and through the vascular wall;27 increase adhesion molecules31 and produce less nitric oxide (NO)26 recruiting more inflammatory cells and reducing vasodilation.32 Hyperglycaemia can also diminish endothelial migration33 and proliferation34 thereby inhibiting angiogenesis in response to injury and ischaemia.35,36 The mechanism of glucoseinduced endothelial cell dysfunction is multifactorial and includes mitochondrial superoxide production, advanced glycation end products (AGE) and protein kinase C (PKC).29 There is now a strong body of evidence to suggest that vascular lesion formation is dependent on a fine balance
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
276
Diabetes & Vascular Disease Research 11(4)
Figure 5. HIF-1α gene silencing inhibits hypoxia-induced changes in BAEC proliferation: (a) and (b) representative scatter data obtained from FACS analysis of CFDA staining for BAEC proliferation in cells transfected with HIF-1α siRNA and scrambled siRNA using the Vybrant® CFDA SE Cell Tracer Kit following exposure of cells to (a) normoxic and (b) hypoxic conditions with or without 25 mM glucose or mannitol; (c) the cumulative cell count data in cells transfected with HIF-1α siRNA and scrambled siRNA following exposure of cells to normoxic and hypoxic conditions with or without 25 mM glucose or mannitol for 5 days (n = 3, #p ≤ 0.01 compared to normoxic scrambled control and *p ≤ 0.01 compared to hypoxic scrambled mannitol). HIF-1α: hypoxia-inducible factor-1α; BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting; CFDA: carboxyfluorescein diacetate; siRNA: small interfering RNA.
between endothelial cell growth and cell death.37–39 The regulation of apoptosis in vascular endothelial cells is complex and consists of multiple interacting pathways within the cell that include those from death receptors and survival cytokines, and the expression of specific intracellular gene products (e.g. those encoded by specific oncogenes, tumour suppressor genes and the Bcl-2 family of genes).40 Vascular homeostasis is clearly compromised by the diabetic state22,41 and by hypoxia;6,7 however, the
precise mechanisms by which these two interactive stimuli may induce vascular dysfunction and promote lesion formation are unclear. Angiogenesis is particularly strongly dependent on the suppression of endothelial cell apoptosis.39 Endothelial cells integrate exogenous pro-angiogenic and anti-angiogenic stimuli and transform them within the cell into conflicting survival and apoptotic signals.39 The prevailing signals may then determine the fate of the endothelial cells and, subsequently, the fate of the growing
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
277
Gao et al.
Figure 6. HIF-1α gene silencing inhibits hypoxia-induced changes in BAEC apoptosis: (a) cumulative data from FACS analysis of BAEC apoptosis in cells transfected with HIF-1α siRNA and scrambled siRNA under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol and (b) caspase-3 enzyme activity in cells transfected with HIF-1α siRNA and scrambled siRNA under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol (n = 3 ± SEM, #p ≤ 0.01 vs normoxic scrambled control, *p ≤ 0.01 vs scrambled and p ≤ 0.01 vs hypoxia siRNA control).
HIF-1α: hypoxia-inducible factor-1α; BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting; siRNA: small interfering RNA; SEM: standard error of mean.
vessel or injured vessel.39 In this study, we demonstrate that hyperglycaemia modulates the growth response of endothelial cells by modulating HIF-1α-dependent expression. This observation as well as our previous work on vascular smooth muscle cells13 highlights a dynamic interplay between the two most important determinants of complications in diabetes, namely, hyperglycaemia and hypoxia, in controlling vascular cell and growth in vitro with important ramifications for vascular health. In this study, glucose alone in the absence of hypoxia was insufficient to activate the HIF-1α pathway. However, it is known that normal glucose concentrations are critical
for HIF-1α protein expression and activation in response to hypoxia since the reduction in normal glucose levels almost completely abolished hypoxic HIF-1α accumulation.14,42 Normal glucose levels (5.5 mM) failed to alter BAEC proliferation or apoptosis over 5 days of normoxia, yet it did facilitate hypoxia-induced HIF-1α expression and HRE-dependent transactivation. However, when endothelial cells were exposed to glucose levels typical of hyperglycaemia, there was reduced HIF-1α expression and HRE-dependent transactivation concomitant with an attenuation of hypoxia-induced changes to BAEC proliferation and apoptosis. In addition, selective knockdown of
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
278
Diabetes & Vascular Disease Research 11(4)
HIF-1α mimicked these effects and blunted the attenuation of hypoxia-induced responses elicited by high glucose. Unlike previous studies on HIF-1α stability,43 we did not observe similar effects of glucose with mannitol while no change to HIF-1α mRNA levels confirmed previous reports that HIF-1α activation is regulated at the protein level.5 To date, several studies have reported reduced levels of HIF-α in tissue from patients with diabetes44 as well as from several animal models of diabetes,45 in conjunction with in vitro studies using cultured cells grown in high glucose medium.13,43 This led to the hypothesis that high glucose levels may contribute to reduced HIF-1α protein levels and transactivation function in diabetes. Several mechanisms have been purported to explain the regulatory effects of high glucose on HIF-1α-mediated responses. Using methylglyoxal (MGO), a highly reactive α-oxoaldehyde and dicarbonyl formed mainly as a by-product of glycolysis and high glucose concentrations, the covalent modification of HIF-α arginine 17 (Arg-17) and arginine 23 (Arg-23) of the bHLH domain (the locus mediating the interaction of HIF-1α with HIF1β) has been demonstrated.46 As a consequence, the impairment of HIF-1α resulted in reduced transcription of HIF target genes which led to defective ischaemiainduced vasculogenesis in diabetic mice.46 Glucoseinduced decreases in HIF-1α also impaired vascular endothelial growth factor (VEGF) production in response to hypoxia, which resulted in reduced neovascularization in cells obtained from diabetic patients and impaired wound healing in ischaemic diabetic animals.47,48 In addition, HIF-1α association with the molecular chaperone heat shock protein 40/70 (Hsp40/70) led to polyubiquitination and proteasomal degradation of HIF-1α. In addition, hyperglycaemia is reported to augment oxidative stress and induce the overproduction of reactive oxygen species (ROS),49 thereby modulating HIF-1α. It − is clear that ROS, in particular superoxide ( O 2 ), can degrade HIF-1α at the post-transcriptional level by activating a proline hydroxylase in the presence of iron and by increasing ubiquitin–proteasome activity.50 NO is also another important regulator of HIF-1α in several cell types.51,52 Moreover, while NO stimulates the accumulation and activation of HIF-1α in endothelial cells,53 − O diminished NO due to glucose-induced 2 accumulation may lead to the inhibition of HIF-1α.52 In this context, we have previously reported that high glucose concentrations attenuated flow and agonist stimulated NO generation in endothelial cells.26 In conclusion, our study in endothelial cells further supports a putative role for glucose in modulating vascular cell growth under hypoxic conditions via a HIF-1α mechanism. This response may have important implications for the tissue’s capacity to adapt to low oxygen tensions under hyperglycaemic conditions.
Declaration of conflicting interests The authors declare that they have no conflicts of interest.
Funding This work was supported in part by funding from the Higher Education Authority (HEA) Programme for Research in Third Level Institutions (PRTLI Cycle III) and in part by funds from Science Foundation Ireland (SFI-11/PI/1128) to P.A.C. and the American Health Foundation (AHF) to C.O.B. and P.A.C.
References 1. Chong ZZ and Maiese K. Mammalian target of rapamycin signaling in diabetic cardiovascular disease. Cardiovasc Diabetol 2012; 11: 45. 2. Peterson LR, McKenzie CR and Schaffer JE. Diabetic cardiovascular disease: getting to the heart of the matter. J Cardiovasc Transl Res 2012; 5: 436–445. 3. Nathan DM, Cleary PA, Backlund J-YC, et al. Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J Med 2005; 353: 2643–2653. 4. Molnar GD, Taylor WF and Langworthy A. On measuring the adequacy of diabetes regulation: comparison of continuously monitored blood glucose patterns with values at selected time points. Diabetologia 1974; 10: 139–143. 5. Nath B and Szabo G. Hypoxia and hypoxia inducible factors: diverse roles in liver diseases. Hepatology 2012; 55: 622–633. 6. Semenza GL. Hypoxia-inducible factor 1 and cardiovascular disease. Annu Rev Physiol 2014; 76: 39–56. 7. Walshe TE and D’Amore PA. The role of hypoxia in vascular injury and repair. Annu Rev Pathol 2008; 3: 615–643. 8. Kim Y-W and Byzova TV. Oxidative stress in angiogenesis and vascular disease. Blood 2014; 123: 625–631. 9. Dötsch J, Knerr I, Rascher W, et al. Does defective hypoxia signalling cause type 2 diabetes? Eur J Endocrinol 2006; 154: 781–782. 10. Gunton JE, Kulkarni RN, Yim S, et al. Loss of ARNT/ HIF1beta mediates altered gene expression and pancreaticislet dysfunction in human type 2 diabetes. Cell 2005; 122: 337–349. 11. Xiao H, Gu Z, Wang G, et al. The possible mechanisms underlying the impairment of HIF-1α pathway signaling in hyperglycemia and the beneficial effects of certain therapies. Int J Med Sci 2013; 10: 1412–1421. 12. Airley RE and Mobasheri A. Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics. Chemotherapy 2007; 53: 233–256. 13. Gao W, Ferguson G, Connell P, et al. High glucose concentrations alter hypoxia-induced control of vascular smooth muscle cell growth via a HIF-1alpha-dependent pathway. J Mol Cell Cardiol 2007; 42: 609–619. 14. Baracca A, Sgarbi G, Padula A, et al. Glucose plays a main role in human fibroblasts adaptation to hypoxia. Int J Biochem Cell Biol 2013; 45: 1356–1365. 15. Colombo A, Guha S, Mackle JN, et al. Cyclic strain amplitude dictates the growth response of vascular smooth muscle cells in vitro: role in in-stent restenosis and inhibition with
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
279
Gao et al. a sirolimus drug-eluting stent. Biomech Model Mechanobiol 2013; 12: 671–683. 16. Sweeney C, Morrow D, Birney YA, et al. Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-Jk dependent pathway. FASEB J 2004; 18: 1421–1423. 17. Romero-Calvo I, Ocón B, Martínez-Moya P, et al. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem 2010; 401: 318–320. 18. Bruick RK. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc Natl Acad Sci USA 2000; 97: 9082–9087. 19. Willard AL and Herman IM. Vascular complications and diabetes: current therapies and future challenges. J Ophthalmol 2012; 2012: 209538 (14 pp.). 20. Pop-Busui R, Sima A and Stevens M. Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 2006; 22: 257–273. 21. Hashmi S and Al-Salam S. Hypoxia-inducible factor-1 alpha in the heart: a double agent? Cardiol Rev 2012; 20: 268–273. 22. Malmberg K, Norhammar A, Wedel H, et al. Glycometabolic state at admission: important risk marker of mortality in conventionally treated patients with diabetes mellitus and acute myocardial infarction: long-term results from the Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study. Circulation 1999; 99: 2626– 2632. 23. Xue W, Cai L, Tan Y, et al. Cardiac-specific overexpression of HIF-1{alpha} prevents deterioration of glycolytic pathway and cardiac remodeling in streptozotocin-induced diabetic mice. Am J Pathol 2010; 177: 97–105. 24. Cioffi CL, Liu XQ, Kosinski PA, et al. Differential regulation of HIF-1 alpha prolyl-4-hydroxylase genes by hypoxia in human cardiovascular cells. Biochem Biophys Res Commun 2003; 303: 947–953. 25. Catrina S-B, Botusan IR, Rantanen A, et al. Hypoxia inducible factor-1alpha and hypoxia-inducible factor-2alpha are expressed in kaposi sarcoma and modulated by insulinlike growth factor-I. Clin Cancer Res 2006; 12: 4506–4514. 26. Connell P, Walshe T, Ferguson G, et al. Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells. Endothelium 2007; 14: 17–24. 27. Hempel A, Maasch C, Heintze U, et al. High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha. Circ Res 1997; 81: 363–371. 28. Joshi M, Kotha SR, Malireddy S, et al. Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria. Mol Cell Biochem 2013; 386: 233–249. 29. Pant R, Marok R and Klein LW. Pathophysiology of coronary vascular remodeling: relationship with traditional risk factors for coronary artery disease. Cardiol Rev 2014; 22: 13–16. 30. Sena CM, Pereira AM and Seiça R. Endothelial dysfunction – a major mediator of diabetic vascular disease. Biochim Biophys Acta 2013; 1832: 2216–2231.
31. Piga R, Naito Y, Kokura S, et al. Short-term high glucose exposure induces monocyte-endothelial cells adhesion and transmigration by increasing VCAM-1 and MCP-1 expression in human aortic endothelial cells. Atherosclerosis 2007; 193: 328–334. 32. Chakravarthy U, Hayes RG, Stitt AW, et al. Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products. Diabetes 1998; 47: 945–952. 33. Hamuro M, Polan J, Natarajan M, et al. High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration. Atherosclerosis 2002; 162: 277– 287. 34. Curcio F and Ceriello A. Decreased cultured endothelial cell proliferation in high glucose medium is reversed by antioxidants: new insights on the pathophysiological mechanisms of diabetic vascular complications. In Vitro Cell Dev Biol 1992; 28A: 787–790. 35. Merchan JR, Kovács K, Railsback JW, et al. Antiangiogenic activity of 2-deoxy-d-glucose. PLoS One 2010; 5: e13699. 36. Martin A, Komada MR and Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev 2003; 23: 117–145. 37. Fisher SA, Langille BL and Srivastava D. Apoptosis during cardiovascular development. Circ Res 200010; 87: 856– 864. 38. Gao C, Sun W, Christofidou-Solomidou M, et al. PECAM-1 functions as a specific and potent inhibitor of mitochondrialdependent apoptosis. Blood 2003; 102: 169–179. 39. Chavakis E and Dimmeler S. Regulation of endothelial cell survival and apoptosis during angiogenesis. Arterioscler Thromb Vasc Biol 2002; 22: 887–893. 40. Ackermann EJ, Taylor JK, Narayana R, et al. The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. J Biol Chem 1999; 274: 11245–11252. 41. Norhammar A, Malmberg K, Diderholm E, et al. Diabetes mellitus: the major risk factor in unstable coronary artery disease even after consideration of the extent of coronary artery disease and benefits of revascularization. J Am Coll Cardiol 2004; 43: 585–591. 42. Staab A, Löffler J, Said HM, et al. Modulation of glucose metabolism inhibits hypoxic accumulation of hypoxiainducible factor-1alpha (HIF-1alpha). Strahlenther Onkol 2007; 183: 366–373. 43. Catrina S-B, Okamoto K, Pereira T, et al. Hyperglycemia regulates hypoxia-inducible factor-1alpha protein stability and function. Diabetes 2004; 53: 3226–3232. 44. Lim JI, Spee C and Hinton DR. A comparison of hypoxiainducible factor-α in surgically excised neovascular membranes of patients with diabetes compared with idiopathic epiretinal membranes in nondiabetic patients. Retina 2010; 30: 1472–1478. 45. Bento CF and Pereira P. Regulation of hypoxia-inducible factor 1 and the loss of the cellular response to hypoxia in diabetes. Diabetologia 2011; 54: 1946–1956. 46. Capla JM, Grogan RH, Callaghan MJ, et al. Diabetes impairs endothelial progenitor cell-mediated blood vessel formation in response to hypoxia. Plast Reconstr Surg 2007; 119: 59–70.
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
280
Diabetes & Vascular Disease Research 11(4)
47. Kajiwara H, Luo Z, Belanger AJ, et al. A hypoxic inducible factor-1 alpha hybrid enhances collateral development and reduces vascular leakage in diabetic rats. J Gene Med 2009; 11: 390–400. 48. Thangarajah H, Yao D, Chang EI, et al. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues. Proc Natl Acad Sci USA 2009; 106: 13505–13510. 49. Baynes JW and Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999; 48: 1–9. 50. Zou A-P and Cowley AW. Reactive oxygen species and molecular regulation of renal oxygenation. Acta Physiol Scand 2003; 179: 233–241.
51. Olson N and Van der Vliet A. Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease. Nitric Oxide 2011; 25: 125–137. 52. Bullock JJ, Mehta SL, Lin Y, et al. Hyperglycemia-enhanced ischemic brain damage in mutant manganese SOD mice is associated with suppression of HIF-1alpha. Neurosci Lett 2009; 456: 89–92. 53. Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000; 95: 189–197.
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
Published by: http://www.sagepublications.com
Additional services and information for Diabetes and Vascular Disease Research can be found at: Email Alerts: http://dvr.sagepub.com/cgi/alerts Subscriptions: http://dvr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Jun 16, 2014 OnlineFirst Version of Record - May 22, 2014 What is This?
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
533356 research-article2014
DVR0010.1177/1479164114533356Diabetes & Vascular Disease ResearchGao et al.
Original Article
Glucose attenuates hypoxia-induced changes in endothelial cell growth by inhibiting HIF-1α expression
Diabetes & Vascular Disease Research 2014, Vol. 11(4) 270–280 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1479164114533356 dvr.sagepub.com
Wei Gao1, Gail Ferguson1, Paul Connell1,2, Tony Walshe1, Colm O’Brien2, Eileen M Redmond3 and Paul A Cahill1
Abstract Hyperglycaemia and hypoxia play essential pathophysiological roles in diabetes. We determined whether hyperglycaemia influences endothelial cell growth under hypoxic conditions in vitro. Using a Ruskinn Invivo2 400 Hypoxia Workstation, bovine aortic endothelial cells (BAEC) were exposed to high glucose concentrations (25 mM glucose) under normoxic or hypoxic conditions before cell growth (balance of proliferation and apoptosis) was assessed by fluorescence-activated cell sorting (FACS) analysis, proliferating cell nuclear antigen (pCNA), Bcl-xL and caspase-3 protein expression and activity. Hypoxia increased hypoxia response element (HRE) transactivation and induced hypoxia-inducible factor-1α (HIF-1α) expression when compared to normoxic controls concomitant with a significant decrease in cell growth. High glucose (25 mM) concentrations attenuated HRE transactivation and HIF-1α protein expression while concurrently reducing hypoxia-induced changes in BAEC growth. Knockdown of HIF-1α expression significantly decreased hypoxiainduced changes in growth and attenuated the modulatory effects of glucose. These results provide evidence that hypoxia-induced control of BAEC growth can be altered by the presence of glucose via inhibition of HIF-1α expression and activation. Keywords Endothelial, hypoxia, hyperglycaemia, HIF-1α, apoptosis, diabetes
Introduction Diabetes mellitus currently affects more than 170 million individuals worldwide and is expected to afflict another 200 million individuals in the next 30 years.1 The majority of diabetic morbidity relates to the incidence of cardiovascular disease where the diabetic patient cohort suffers from accelerated and severe atherosclerosis that can lead to heart attack, stroke and peripheral vascular disease.2 While diabetes is an established risk factor for vascular disease such as atherosclerosis, the mechanism by which diabetes accelerates the disease remains unknown. Glucose fluctuations characteristic of both Type I and Type II diabetes have been implicated in diabetic atherosclerosis as tight glycaemic control has been shown to significantly reduce the risk of myocardial infarction and stroke in people with diabetes.3,4 Hypoxia is a reduction in oxygen delivery below tissue demand and can be either acute or chronic. It is centrally regulated by hypoxia-inducible factor (HIF), a heterodimer composed of α and β subunits that belong to the basic helix–loop–helix (bHLH) Per, Arnt, Sim (PAS) family of transcription factors.5,6 While severe chronic hypoxia can
cause cell death, more modest hypoxia can protect against subsequent cell damage.7 The intensity and sustainability of HIF-1α activation are considered one of the major determinants of whether responses are pathological or beneficial. Several important processes are characterized by hypoxia, including ischaemia–reperfusion, tumour growth and progression, inflammation, myocardial ischaemia and a number of ocular pathologies.7,8
1Vascular
Biology and Therapeutics Laboratory, School of Biotechnology, Faculty of Science and Health, Dublin City University, Dublin, Ireland 2Mater Misericordiae Hospital, Institute of Ophthalmology, The Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland 3Department of Surgery, University of Rochester, Rochester, NY, USA Corresponding author: Paul A Cahill, Vascular Biology and Therapeutics Laboratory, School of Biotechnology, Faculty of Science and Health, Dublin City University, Dublin 9, Ireland. Email: [email protected]
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
271
Gao et al. There have been numerous studies demonstrating reduced levels of HIF-α from patients with diabetes and animal models of diabetes in conjunction with mammalian cells cultured in high glucose medium.9–11 Hyperglycaemia appears to compromise HIF-α protein levels and transactivation function, yet the detailed molecular mechanisms underlying impairment of HIF-α by high glucose remain poorly understood. It is known that HIF-1α regulates the expression of enzymes involved in the process of glycolysis and of GLUT1 and GLUT3 which mediate cellular glucose uptake.12 On the other hand, glucose, glucose uptake and glycolysis influence the stability and activation of HIF-1α.13,14 In this study, we examined the effects of high glucose levels on endothelial cell growth (the balance of proliferation and apoptosis) under normoxic and hypoxic conditions while investigating the putative role of HIF-1α in this response. Our data provide evidence that hypoxia-induced changes in endothelial cell growth can be attenuated by glucose through modulation of HIF-1α.
Methods Materials All materials were of the highest purity and were purchased from Sigma–Aldrich unless otherwise stated. Antibodies were purchased from the vendors for proliferating cell nuclear antigen (pCNA) (1:80; Cayman Chemical Co., Ann Arbor, MI, USA), caspase-3 (1:1000; Cell Signaling Technology, MSC, Ireland), Bcl-xL (1:200; BD Biosciences Europe, UK), HIF-1α (1:1000; BD Biosciences) or BNIP3L [1:500; Axxora (UK) Ltd].
Cell culture Bovine aortic endothelial cells (BAEC) were purchased from the Coriell Cell Repositories (Cat AG07680) and grown in normal glucose (control: 5.5 mM) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. Cell lines were maintained in a humidified atmosphere of 5% CO2/95% air (normoxic conditions) and routinely used between passages 9 and 14.
Hypoxic/high-glucose studies Cells were quiesced for 48 h in control DMEM supplemented with 0.5% FBS before cells were exposed to normoxic (5% CO2, 95% air) or chronic hypoxic (2% O2, 5% CO2, 93% N2) conditions in DMEM supplemented with 10% FBS in the presence of normal glucose (C: control: 5.5 mM) or high glucose (G: glucose: 25 mM) or a mannitol control (M: mannitol 25 mM) for 5 days. Media were replaced daily using the Ruskinn Invivo2 400 Hypoxia Workstation as previously described.13
Proliferation and apoptosis assays Effects on cell proliferation and apoptosis were determined using the Vybrant® CFDA SE Cell Proliferation Kit and the Vybrant Apoptosis Assay Kit #2 (Molecular Probes®, Invitrogen, Ireland) followed by fluorescence-activated cell sorting (FACS) analysis using a FACSCAN flow cytometer (Becton, Dickinson and Company, Oxford UK) as previously described.13,15 Apoptosis was also determined by measuring caspase-3 activity and expression using an assay based on the cleavage of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) and an anti-caspase-3 antibody, respectively, as previously described by us.13
Western blotting Soluble extracts (15–40 µg) were fractionated by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) on 6%–12% (w/v) polyacrylamide resolving gels depending on protein size to be detected as previously described.13 Scanning densitometry was performed with image analysis software [one-dimensional (1D); Eastman-Kodak, Rochester, NY, USA]. Minor differences in protein loading and transfer were normalized using a Ponceau S stain as previously described16 and validated as before.17
Semi-quantitative reverse transcriptionpolymerase chain reaction Total RNA was isolated from cells using TRIzol™ (Invitrogen) according to the manufacturer’s specifications and was reverse transcribed to complementary DNA (cDNA) using iScript as per the manufacturer’s specifications (Alpha Technologies, UK) as previously described.13
Transient transfections BAEC were plated in six-well plates and incubated for 3 h in 1 mL/well DMEM containing 2 µg of cDNA or 100 µg/ mL scrambled or small interfering RNA (siRNA)-specific duplexes and 4 µL of Lipofectamine™ (Invitrogen™, BioSciences Ltd, Ireland) according to the manufacturer’s instructions. The siRNA duplexes for HIF-1α corresponded to positions 1254–1274 in the bovine HIF-1α sequence (accession number: NM_174339) and were acquired from M W G Biotech (UK). Scrambled siRNA duplexes were used as controls. The hypoxia response element (HRE)-luciferase reporter plasmid was a kind gift from Dr Cormac Taylor, Conway Institute of Biomolecular & Biomedical Research, Ireland. DNA transfection efficiency was confirmed and normalized to β-galactosidase activity following co-transfection with CMV-LacZ, a plasmid-encoding β-galactosidase activity as previously described.13
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
272
Diabetes & Vascular Disease Research 11(4)
Statistical analysis All treatments were normalized to quiesced cells (serumdeprived control at day 0) and expressed as a fold increase or decrease compared to this value. The results are expressed as mean ± standard error of mean (SEM). Statistical significance was assessed by analysis of variance (ANOVA) with significance values of p ≤ 0.01 and p ≤ 0.05 for difference between groups.
Results BAEC were initially grown in control DMEM containing glucose (5.5 mM) before the cells were exposed to normoxic or hypoxic conditions for 5 days. HRE-luciferase activity was negligible under normoxic conditions. However, following exposure to hypoxia, HRE transactivation increased significantly, an effect that was significantly attenuated with glucose (25 mM) (Figure 1(a)). In parallel cultures, HIF-1α protein subunit levels were undetectable after 5 days under normoxic conditions (Figure 1(b)) but increased dramatically after 5 days under hypoxic conditions (Figure 1(b)). HIF-1α protein expression was significantly attenuated, without any significant change in HIF-1α messenger RNA (mRNA) levels, when cells were exposed to hypoxia in the presence of glucose (25 mM) (Figure 1(b) and (c)). The effect of hypoxia and hyperglycaemia on BAEC proliferation was initially examined by FACS analysis using a carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) fluorescent cell assay. Exposure of cells to 0.5% and 10% foetal calf serum (FCS) for 5 days confirmed that a shift to the left in the CFDA FACS scatter plot represented a significant increase in cell proliferation (data not shown). In response to hypoxia, there was a dramatic shift to the right representing a decrease in cell proliferation when compared to normoxia (Figure 2(a)). While glucose (25 mM) did not alter the proliferation state of the cells under normoxia (Figure 2(b)), it did result in a significant increase in cell proliferation following hypoxia (Figure 2(c)). Parallel studies confirmed that the number of cells significantly increased at day 5 when compared to C (t = 0). Moreover, under normoxic conditions, the number of cells after 5 days was similar following treatment with high glucose (G: 25 mM) when compared to control glucose (C: 5.5 mM) or mannitol (M: 25 mM) treatment (Figure 2(d)). In contrast, hypoxia resulted in a significant decrease in cell number, an effect that was attenuated following exposure to glucose (25 mM) (Figure 2(d)). Similarly, the level of pCNA expression, a cyclin marker of cell proliferation, was increased after 5 days under normoxia when compared to day 0 but was unaffected following treatment with glucose. However, hypoxia significantly decreased pCNA expression, an effect that was attenuated by the addition of glucose (Figure 2(e)).
Figure 1. Effect of hyperglycaemia and hypoxia on HIF-1α activity and expression: (a) hypoxia-induced HRE-luciferase transactivation under normoxic and hypoxic conditions (n = 3 ± SEM, #p ≤ 0.01 compared to normoxic control, *p ≤ 0.01 compared to hypoxia mannitol); (b) representative immunoblot and cumulative data of HIF-1α expression in BAEC under normoxic and hypoxic conditions (n = 3 ± SEM, *p ≤ 0.01 compared to mannitol) and (c) representative qRT-PCR of HIF-1α mRNA levels in BAEC under normoxic and hypoxic conditions [C, control (5.5 mM glucose), G (25 mM glucose) and M (25 mM mannitol)].
HIF-1α: hypoxia-inducible factor-1α; HRE: hypoxia response element; SEM: standard error of mean; BAEC: bovine aortic endothelial cells; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; mRNA: messenger RNA.
As growth is the balance of proliferation and apoptosis, we also examined the effect of hypoxia on BAEC apoptosis in the absence or presence of glucose. There was no significant change in apoptosis following treatment of
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
273
Gao et al.
Figure 2. Effect of hyperglycaemia on hypoxia-induced changes in BAEC proliferation: (a) representative scatter data obtained from FACS analysis of CFDA staining for BAEC proliferation at days 0 and 5 under normoxic and hypoxic conditions using the Vybrant® CFDA SE Cell Tracer Kit. Representative scatter data obtained from FACS analysis of BAEC proliferation under (b) normoxic and (c) hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol. (d) Cumulative cell count data of BAEC proliferation under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol and (e) Western blot analysis of concurrent cell lysates for pCNA in the presence or absence of glucose and mannitol (25 mM). Data are the mean ± SEM for three independent experiments performed in triplicate (#p ≤ 0.01 compared to day 0, *p ≤ 0.01 compared to normoxic control and ¶p ≤ 0.01 compared to hypoxia mannitol) [C (t = 0) = quiesced cells at time 0: prior to treatment; C = 10% FBS media; G = 10% FBS media + 25 mM glucose; M = 10% FBS media + 25 mM mannitol]. BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting; CFDA: carboxyfluorescein diacetate; pCNA: proliferating cell nuclear antigen; SEM: standard error of mean; FBS: foetal bovine serum.
cells with high glucose levels (25 mM) under normoxia (Figure 3(a)). In contrast, hypoxia promoted apoptosis, an effect that was reduced in the presence of glucose (Figure 3(a)). Further analysis of apoptosis by colorimetric measurement of caspase-3 activity and immunoblot detection of caspase-3 protein levels confirmed that the hypoxiainduced changes in caspase-3 activity and protein expression were attenuated in the presence of glucose (Figure 3(b) and (c)).
The effects of hypoxia on the stress activator response18 were also investigated. Hypoxia caused a reduction in the expression of the anti-apoptotic protein, Bcl-xL, when compared to normoxia (Figure 3(d)), without any change in Bcl-xL mRNA levels (data not shown). However, the decrease in Bcl-xL protein expression was attenuated by glucose (25 mM) (Figure 3(d)), again without any change in Bcl-xL mRNA levels (data not shown). Further analysis of the expression of the pro-apoptotic protein BNIP3L, a
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
274
Diabetes & Vascular Disease Research 11(4)
Figure 3. Effect of hyperglycaemia on hypoxia-induced changes in BAEC apoptosis: (a) representative data obtained from FACS analysis of BAEC apoptosis using the Vybrant® Apoptosis Assay Kit (inset: V = viable, EA = early apoptotic, LA = late apoptotic, N = necrotic). The values obtained for EA and LA cells were combined and plotted as a bar chart: (b) caspase-3 enzyme activity and (c) representative immunoblot of caspase-3 expression under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol, and (d) effect of hyperglycaemia and hypoxia on Bcl-xL expression in BAEC. Representative immunoblot and cumulative data of Bcl-xL and BNIP3L expression in BAEC under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and mannitol (n = 3, #p ≤ 0.01 compared to normoxic control, *p ≤ 0.01 compared to hypoxic mannitol). BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting.
natural suppressor of Bcl-xL activity, confirmed that hypoxia promoted BNIP3L protein expression (Figure 3(d)), a response that was significantly reduced by glucose (Figure 3(d)). The putative role of HIF-1α in regulating hypoxiainduced changes in BAEC growth (balance of proliferation and apoptosis) was examined in cells following HIF-1α knockdown. Initial quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis
confirmed HIF-1α knockdown (Figure 4(a)). BAEC proliferation remained unchanged in cells transfected with either HIF-1α or scrambled siRNA under normoxia when cells were treated with glucose (25 mM) (Figure 5(a)). In contrast, hypoxia inhibited BAEC proliferation in cells transfected with scrambled siRNA (Figure 5(b) vs 5(a)), an effect that was attenuated by glucose (Figure 5(b)). However, this recovery in BAEC proliferation by glucose (25 mM) was abolished following HIF-1α knockdown (Figure 5(b)). In
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
275
Gao et al.
transfected with scrambled siRNA (Figure 6(a)), an effect that was significantly reduced following HIF-1α knockdown (Figure 6(a)). Furthermore, the level of apoptosis was unchanged following treatment of cells with glucose (Figure 6(a)). In parallel cultures, caspase-3 activity was also unchanged following scrambled siRNA or HIF-1α knockdown under normoxia (Figure 6(b)) with or without glucose (Figure 6(b)). However, hypoxia increased caspase-3 activity in scrambled siRNA-transfected cells (Figure 6(b)), an effect that was significantly reduced following knockdown of HIF-1α (Figure 6(b)). Moreover, caspase-3 activity was not significantly further reduced following treatment of cells with glucose (Figure 6(b)).
Discussion
Figure 4. HIF-1α gene silencing inhibits hypoxia-induced HIF-1α: (a) HIF-1α mRNA levels in cells transfected with HIF1α siRNA compared to scrambled and NT controls [n = 3 ± SEM, *p ≤ 0.01 compared to scrambled (Scram) control] and (b) HIF-1α protein expression in cells transfected with HIF-1α siRNA compared to scrambled and NT controls [n = 3 ± SEM, *p ≤ 0.01 compared to scrambled (Scram) control].
HIF-1α: hypoxia-inducible factor-1α; siRNA: small interfering RNA; NT: non-transfected; SEM: standard error of mean; mRNA: messenger RNA.
parallel studies, hypoxia promoted a decrease in cell number in scrambled controls, an effect that was attenuated by glucose. However, when cells were transfected with siRNA against HIF-1α, there was no significant change in cell number under normoxia or hypoxia (Figure 5(c)). In addition, there was no significant difference in cell number following treatment with glucose (Figure 5(c)). HIF-1α knockdown resulted in a modest reduction in the baseline level of apoptosis under normoxia (Figure 6(a)). There was no significant effect of glucose (Figure 6(a)). In contrast, hypoxia promoted apoptosis in BAEC
The causative factors of diabetic retinopathy,19 neuropathy20 and arteriosclerosis6,21 include hyperglycaemia and hypoxia. This is primarily because blood glucose is strongly associated with morbidity after an acute hypoxic challenge, that is, acute myocardial infarction, suggesting a potential deleterious influence of hyperglycaemia on the tissue’s capacity to adapt to low oxygen tensions.22 As a result, there exists a close interrelationship between glucose and HIF-1α in several biological systems. HIF-1α regulates the expression of numerous enzymes involved in the process of glycolysis,12 whereas glucose uptake and glycolysis can significantly influence the stability and activation of HIF-1α.6,8,23 Hypoxia-induced HIF-1α activation is very well characterized for numerous cell types,24 including vascular endothelial cells.7 Glucose has also been shown to affect HIF-1α expression and activity in several cell types11,25 including vascular smooth muscle cells.13 We therefore assessed the effect of hyperglycaemia on HIF-1α expression and activity in endothelial cells. Several studies have demonstrated that altered glucose levels contribute to the endothelial cell dysfunction,26–28 which is a major initiating step in vascular disease.29 Healthy endothelial cells maintain vascular homeostasis through tight control of permeability, inflammation, vascular tone and injury repair responses.30 However, high glucose levels can promote increased endothelial cell permeability, allowing solutes to pass into and through the vascular wall;27 increase adhesion molecules31 and produce less nitric oxide (NO)26 recruiting more inflammatory cells and reducing vasodilation.32 Hyperglycaemia can also diminish endothelial migration33 and proliferation34 thereby inhibiting angiogenesis in response to injury and ischaemia.35,36 The mechanism of glucoseinduced endothelial cell dysfunction is multifactorial and includes mitochondrial superoxide production, advanced glycation end products (AGE) and protein kinase C (PKC).29 There is now a strong body of evidence to suggest that vascular lesion formation is dependent on a fine balance
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
276
Diabetes & Vascular Disease Research 11(4)
Figure 5. HIF-1α gene silencing inhibits hypoxia-induced changes in BAEC proliferation: (a) and (b) representative scatter data obtained from FACS analysis of CFDA staining for BAEC proliferation in cells transfected with HIF-1α siRNA and scrambled siRNA using the Vybrant® CFDA SE Cell Tracer Kit following exposure of cells to (a) normoxic and (b) hypoxic conditions with or without 25 mM glucose or mannitol; (c) the cumulative cell count data in cells transfected with HIF-1α siRNA and scrambled siRNA following exposure of cells to normoxic and hypoxic conditions with or without 25 mM glucose or mannitol for 5 days (n = 3, #p ≤ 0.01 compared to normoxic scrambled control and *p ≤ 0.01 compared to hypoxic scrambled mannitol). HIF-1α: hypoxia-inducible factor-1α; BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting; CFDA: carboxyfluorescein diacetate; siRNA: small interfering RNA.
between endothelial cell growth and cell death.37–39 The regulation of apoptosis in vascular endothelial cells is complex and consists of multiple interacting pathways within the cell that include those from death receptors and survival cytokines, and the expression of specific intracellular gene products (e.g. those encoded by specific oncogenes, tumour suppressor genes and the Bcl-2 family of genes).40 Vascular homeostasis is clearly compromised by the diabetic state22,41 and by hypoxia;6,7 however, the
precise mechanisms by which these two interactive stimuli may induce vascular dysfunction and promote lesion formation are unclear. Angiogenesis is particularly strongly dependent on the suppression of endothelial cell apoptosis.39 Endothelial cells integrate exogenous pro-angiogenic and anti-angiogenic stimuli and transform them within the cell into conflicting survival and apoptotic signals.39 The prevailing signals may then determine the fate of the endothelial cells and, subsequently, the fate of the growing
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
277
Gao et al.
Figure 6. HIF-1α gene silencing inhibits hypoxia-induced changes in BAEC apoptosis: (a) cumulative data from FACS analysis of BAEC apoptosis in cells transfected with HIF-1α siRNA and scrambled siRNA under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol and (b) caspase-3 enzyme activity in cells transfected with HIF-1α siRNA and scrambled siRNA under normoxic and hypoxic conditions in the absence or presence of 25 mM glucose and 25 mM mannitol (n = 3 ± SEM, #p ≤ 0.01 vs normoxic scrambled control, *p ≤ 0.01 vs scrambled and p ≤ 0.01 vs hypoxia siRNA control).
HIF-1α: hypoxia-inducible factor-1α; BAEC: bovine aortic endothelial cells; FACS: fluorescence-activated cell sorting; siRNA: small interfering RNA; SEM: standard error of mean.
vessel or injured vessel.39 In this study, we demonstrate that hyperglycaemia modulates the growth response of endothelial cells by modulating HIF-1α-dependent expression. This observation as well as our previous work on vascular smooth muscle cells13 highlights a dynamic interplay between the two most important determinants of complications in diabetes, namely, hyperglycaemia and hypoxia, in controlling vascular cell and growth in vitro with important ramifications for vascular health. In this study, glucose alone in the absence of hypoxia was insufficient to activate the HIF-1α pathway. However, it is known that normal glucose concentrations are critical
for HIF-1α protein expression and activation in response to hypoxia since the reduction in normal glucose levels almost completely abolished hypoxic HIF-1α accumulation.14,42 Normal glucose levels (5.5 mM) failed to alter BAEC proliferation or apoptosis over 5 days of normoxia, yet it did facilitate hypoxia-induced HIF-1α expression and HRE-dependent transactivation. However, when endothelial cells were exposed to glucose levels typical of hyperglycaemia, there was reduced HIF-1α expression and HRE-dependent transactivation concomitant with an attenuation of hypoxia-induced changes to BAEC proliferation and apoptosis. In addition, selective knockdown of
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
278
Diabetes & Vascular Disease Research 11(4)
HIF-1α mimicked these effects and blunted the attenuation of hypoxia-induced responses elicited by high glucose. Unlike previous studies on HIF-1α stability,43 we did not observe similar effects of glucose with mannitol while no change to HIF-1α mRNA levels confirmed previous reports that HIF-1α activation is regulated at the protein level.5 To date, several studies have reported reduced levels of HIF-α in tissue from patients with diabetes44 as well as from several animal models of diabetes,45 in conjunction with in vitro studies using cultured cells grown in high glucose medium.13,43 This led to the hypothesis that high glucose levels may contribute to reduced HIF-1α protein levels and transactivation function in diabetes. Several mechanisms have been purported to explain the regulatory effects of high glucose on HIF-1α-mediated responses. Using methylglyoxal (MGO), a highly reactive α-oxoaldehyde and dicarbonyl formed mainly as a by-product of glycolysis and high glucose concentrations, the covalent modification of HIF-α arginine 17 (Arg-17) and arginine 23 (Arg-23) of the bHLH domain (the locus mediating the interaction of HIF-1α with HIF1β) has been demonstrated.46 As a consequence, the impairment of HIF-1α resulted in reduced transcription of HIF target genes which led to defective ischaemiainduced vasculogenesis in diabetic mice.46 Glucoseinduced decreases in HIF-1α also impaired vascular endothelial growth factor (VEGF) production in response to hypoxia, which resulted in reduced neovascularization in cells obtained from diabetic patients and impaired wound healing in ischaemic diabetic animals.47,48 In addition, HIF-1α association with the molecular chaperone heat shock protein 40/70 (Hsp40/70) led to polyubiquitination and proteasomal degradation of HIF-1α. In addition, hyperglycaemia is reported to augment oxidative stress and induce the overproduction of reactive oxygen species (ROS),49 thereby modulating HIF-1α. It − is clear that ROS, in particular superoxide ( O 2 ), can degrade HIF-1α at the post-transcriptional level by activating a proline hydroxylase in the presence of iron and by increasing ubiquitin–proteasome activity.50 NO is also another important regulator of HIF-1α in several cell types.51,52 Moreover, while NO stimulates the accumulation and activation of HIF-1α in endothelial cells,53 − O diminished NO due to glucose-induced 2 accumulation may lead to the inhibition of HIF-1α.52 In this context, we have previously reported that high glucose concentrations attenuated flow and agonist stimulated NO generation in endothelial cells.26 In conclusion, our study in endothelial cells further supports a putative role for glucose in modulating vascular cell growth under hypoxic conditions via a HIF-1α mechanism. This response may have important implications for the tissue’s capacity to adapt to low oxygen tensions under hyperglycaemic conditions.
Declaration of conflicting interests The authors declare that they have no conflicts of interest.
Funding This work was supported in part by funding from the Higher Education Authority (HEA) Programme for Research in Third Level Institutions (PRTLI Cycle III) and in part by funds from Science Foundation Ireland (SFI-11/PI/1128) to P.A.C. and the American Health Foundation (AHF) to C.O.B. and P.A.C.
References 1. Chong ZZ and Maiese K. Mammalian target of rapamycin signaling in diabetic cardiovascular disease. Cardiovasc Diabetol 2012; 11: 45. 2. Peterson LR, McKenzie CR and Schaffer JE. Diabetic cardiovascular disease: getting to the heart of the matter. J Cardiovasc Transl Res 2012; 5: 436–445. 3. Nathan DM, Cleary PA, Backlund J-YC, et al. Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J Med 2005; 353: 2643–2653. 4. Molnar GD, Taylor WF and Langworthy A. On measuring the adequacy of diabetes regulation: comparison of continuously monitored blood glucose patterns with values at selected time points. Diabetologia 1974; 10: 139–143. 5. Nath B and Szabo G. Hypoxia and hypoxia inducible factors: diverse roles in liver diseases. Hepatology 2012; 55: 622–633. 6. Semenza GL. Hypoxia-inducible factor 1 and cardiovascular disease. Annu Rev Physiol 2014; 76: 39–56. 7. Walshe TE and D’Amore PA. The role of hypoxia in vascular injury and repair. Annu Rev Pathol 2008; 3: 615–643. 8. Kim Y-W and Byzova TV. Oxidative stress in angiogenesis and vascular disease. Blood 2014; 123: 625–631. 9. Dötsch J, Knerr I, Rascher W, et al. Does defective hypoxia signalling cause type 2 diabetes? Eur J Endocrinol 2006; 154: 781–782. 10. Gunton JE, Kulkarni RN, Yim S, et al. Loss of ARNT/ HIF1beta mediates altered gene expression and pancreaticislet dysfunction in human type 2 diabetes. Cell 2005; 122: 337–349. 11. Xiao H, Gu Z, Wang G, et al. The possible mechanisms underlying the impairment of HIF-1α pathway signaling in hyperglycemia and the beneficial effects of certain therapies. Int J Med Sci 2013; 10: 1412–1421. 12. Airley RE and Mobasheri A. Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics. Chemotherapy 2007; 53: 233–256. 13. Gao W, Ferguson G, Connell P, et al. High glucose concentrations alter hypoxia-induced control of vascular smooth muscle cell growth via a HIF-1alpha-dependent pathway. J Mol Cell Cardiol 2007; 42: 609–619. 14. Baracca A, Sgarbi G, Padula A, et al. Glucose plays a main role in human fibroblasts adaptation to hypoxia. Int J Biochem Cell Biol 2013; 45: 1356–1365. 15. Colombo A, Guha S, Mackle JN, et al. Cyclic strain amplitude dictates the growth response of vascular smooth muscle cells in vitro: role in in-stent restenosis and inhibition with
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
279
Gao et al. a sirolimus drug-eluting stent. Biomech Model Mechanobiol 2013; 12: 671–683. 16. Sweeney C, Morrow D, Birney YA, et al. Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-Jk dependent pathway. FASEB J 2004; 18: 1421–1423. 17. Romero-Calvo I, Ocón B, Martínez-Moya P, et al. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem 2010; 401: 318–320. 18. Bruick RK. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc Natl Acad Sci USA 2000; 97: 9082–9087. 19. Willard AL and Herman IM. Vascular complications and diabetes: current therapies and future challenges. J Ophthalmol 2012; 2012: 209538 (14 pp.). 20. Pop-Busui R, Sima A and Stevens M. Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 2006; 22: 257–273. 21. Hashmi S and Al-Salam S. Hypoxia-inducible factor-1 alpha in the heart: a double agent? Cardiol Rev 2012; 20: 268–273. 22. Malmberg K, Norhammar A, Wedel H, et al. Glycometabolic state at admission: important risk marker of mortality in conventionally treated patients with diabetes mellitus and acute myocardial infarction: long-term results from the Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study. Circulation 1999; 99: 2626– 2632. 23. Xue W, Cai L, Tan Y, et al. Cardiac-specific overexpression of HIF-1{alpha} prevents deterioration of glycolytic pathway and cardiac remodeling in streptozotocin-induced diabetic mice. Am J Pathol 2010; 177: 97–105. 24. Cioffi CL, Liu XQ, Kosinski PA, et al. Differential regulation of HIF-1 alpha prolyl-4-hydroxylase genes by hypoxia in human cardiovascular cells. Biochem Biophys Res Commun 2003; 303: 947–953. 25. Catrina S-B, Botusan IR, Rantanen A, et al. Hypoxia inducible factor-1alpha and hypoxia-inducible factor-2alpha are expressed in kaposi sarcoma and modulated by insulinlike growth factor-I. Clin Cancer Res 2006; 12: 4506–4514. 26. Connell P, Walshe T, Ferguson G, et al. Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells. Endothelium 2007; 14: 17–24. 27. Hempel A, Maasch C, Heintze U, et al. High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha. Circ Res 1997; 81: 363–371. 28. Joshi M, Kotha SR, Malireddy S, et al. Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria. Mol Cell Biochem 2013; 386: 233–249. 29. Pant R, Marok R and Klein LW. Pathophysiology of coronary vascular remodeling: relationship with traditional risk factors for coronary artery disease. Cardiol Rev 2014; 22: 13–16. 30. Sena CM, Pereira AM and Seiça R. Endothelial dysfunction – a major mediator of diabetic vascular disease. Biochim Biophys Acta 2013; 1832: 2216–2231.
31. Piga R, Naito Y, Kokura S, et al. Short-term high glucose exposure induces monocyte-endothelial cells adhesion and transmigration by increasing VCAM-1 and MCP-1 expression in human aortic endothelial cells. Atherosclerosis 2007; 193: 328–334. 32. Chakravarthy U, Hayes RG, Stitt AW, et al. Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products. Diabetes 1998; 47: 945–952. 33. Hamuro M, Polan J, Natarajan M, et al. High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration. Atherosclerosis 2002; 162: 277– 287. 34. Curcio F and Ceriello A. Decreased cultured endothelial cell proliferation in high glucose medium is reversed by antioxidants: new insights on the pathophysiological mechanisms of diabetic vascular complications. In Vitro Cell Dev Biol 1992; 28A: 787–790. 35. Merchan JR, Kovács K, Railsback JW, et al. Antiangiogenic activity of 2-deoxy-d-glucose. PLoS One 2010; 5: e13699. 36. Martin A, Komada MR and Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev 2003; 23: 117–145. 37. Fisher SA, Langille BL and Srivastava D. Apoptosis during cardiovascular development. Circ Res 200010; 87: 856– 864. 38. Gao C, Sun W, Christofidou-Solomidou M, et al. PECAM-1 functions as a specific and potent inhibitor of mitochondrialdependent apoptosis. Blood 2003; 102: 169–179. 39. Chavakis E and Dimmeler S. Regulation of endothelial cell survival and apoptosis during angiogenesis. Arterioscler Thromb Vasc Biol 2002; 22: 887–893. 40. Ackermann EJ, Taylor JK, Narayana R, et al. The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. J Biol Chem 1999; 274: 11245–11252. 41. Norhammar A, Malmberg K, Diderholm E, et al. Diabetes mellitus: the major risk factor in unstable coronary artery disease even after consideration of the extent of coronary artery disease and benefits of revascularization. J Am Coll Cardiol 2004; 43: 585–591. 42. Staab A, Löffler J, Said HM, et al. Modulation of glucose metabolism inhibits hypoxic accumulation of hypoxiainducible factor-1alpha (HIF-1alpha). Strahlenther Onkol 2007; 183: 366–373. 43. Catrina S-B, Okamoto K, Pereira T, et al. Hyperglycemia regulates hypoxia-inducible factor-1alpha protein stability and function. Diabetes 2004; 53: 3226–3232. 44. Lim JI, Spee C and Hinton DR. A comparison of hypoxiainducible factor-α in surgically excised neovascular membranes of patients with diabetes compared with idiopathic epiretinal membranes in nondiabetic patients. Retina 2010; 30: 1472–1478. 45. Bento CF and Pereira P. Regulation of hypoxia-inducible factor 1 and the loss of the cellular response to hypoxia in diabetes. Diabetologia 2011; 54: 1946–1956. 46. Capla JM, Grogan RH, Callaghan MJ, et al. Diabetes impairs endothelial progenitor cell-mediated blood vessel formation in response to hypoxia. Plast Reconstr Surg 2007; 119: 59–70.
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
280
Diabetes & Vascular Disease Research 11(4)
47. Kajiwara H, Luo Z, Belanger AJ, et al. A hypoxic inducible factor-1 alpha hybrid enhances collateral development and reduces vascular leakage in diabetic rats. J Gene Med 2009; 11: 390–400. 48. Thangarajah H, Yao D, Chang EI, et al. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues. Proc Natl Acad Sci USA 2009; 106: 13505–13510. 49. Baynes JW and Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999; 48: 1–9. 50. Zou A-P and Cowley AW. Reactive oxygen species and molecular regulation of renal oxygenation. Acta Physiol Scand 2003; 179: 233–241.
51. Olson N and Van der Vliet A. Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease. Nitric Oxide 2011; 25: 125–137. 52. Bullock JJ, Mehta SL, Lin Y, et al. Hyperglycemia-enhanced ischemic brain damage in mutant manganese SOD mice is associated with suppression of HIF-1alpha. Neurosci Lett 2009; 456: 89–92. 53. Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000; 95: 189–197.
Downloaded from dvr.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on November 24, 2014
