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HDL induces anergy to proinflammatory cytokines in endothelial cells through a mild oxidative stress response
Abstracts
S243
years. 54% were men, and 15% were current smokers at baseline. The 4-year predicted risk of CHD was overestimated by 198% (95% confidence interval [95% CI]: 162–238%) with UKPDS, and by 146% (117–179%) and 289% (243–341%) using two different Framingham equations. The area under the receiver-operating curves characteristics (95% CI) for the UKPDS equation was 0.692 (0.660–0.723), and 0.650 (0.616–0.684) and 0.666 (0.634–0.699) for the Framingham equations. Within categories of predicted probabilities, the three equations showed a significant lack of fit, with always a systematic overestimation of the risks. The results were consistent for men and women, Caucasians and non-Caucasians. Conclusions: Uncritical application of the Framingham and UKPDS risk function to a contemporary population with diabetes is likely to overestimate coronary risk, which has important implications for cardiovascular preventative strategies. Enhanced risk prediction tools are needed to reliably tailor such strategies in people with diabetes.
impairment of blood flow recovery following hindlimb ischaemia (0.336 ± 0.034 vs. 0.525 ± 0.021; p < 0.0002). However, i.m. injection of Txnip siRNA dramatically increased limb perfusion in diabetic mice to levels nearing control (0.443 ± 0047; p = 0.153). Capillary density was significantly increased by Txnip siRNA (2.7 ± 0.8-fold), while ischaemia (0.0 ± 0.0 vs. 0.5 ± 0.1) and foot movement scores (0.2 ± 0.1 vs. 0.8 ± 0.2) also significantly improved (p < 0.04). Conclusions: The TRX/TXNIP system plays a key role in angiogenesis and impaired neovascularisation in DM. Our findings demonstrate that inhibition of TXNIP can abrogate the deleterious effects of hyperglycaemia on angiogenesis.
doi:10.1016/j.hlc.2009.05.600
A.K. Heather 1,2,3, , X.H. Li 1,2,3 , K.C.Y. McGrath 1,2,3 , K.A. Rye 1,2,3 , P.J. Barter 1,2,3
555 GENE SILENCING OF THIOREDOXIN INTERACTING PROTEIN DRAMATICALLY RESCUES DIABETESRELATED IMPAIRMENT OF ANGIOGENESIS IN VITRO AND IN VIVO L.L. Dunn 1 , A.M. Buckle 1 , P. Simpson 1 , R.W.Y. Chow 1 , D. Sieveking 1 , M.K. Ng 1,2 1 Heart
Research Institute, Sydney, Australia of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia
2 Department
Background: We recently identified a role for thioredoxin (TRX) and its endogenous inhibitor, thioredoxin interacting protein (TXNIP) in angiogenesis. As TXNIP is highly induced by glucose, we hypothesise that the TXNIP/TRX system is dysregulated in diabetes mellitus (DM) leading to impaired angiogenesis. Methods: (i) Human umbilical vein endothelial cells (HUVECs) were cultured in 5–25 mM glucose and siRNA utilised to modulate TXNIP. Angiogenic assays included: cell proliferation, migration, Matrigel tubulogenesis, TRX activity and VEGF ELISA. (ii) Unilateral hindlimb ischaemia was introduced in a murine model of streptozotocin-induced DM. Plasmid encoding Txnip siRNA or scrambled control was injected intramuscularly (i.m.) and limb perfusion assessed by Laser Doppler. Ischaemia, foot movement and capillary density were assessed. Results: Hyperglycaemia (25 mM) induced TXNIP in HUVECs (5.1 ± 1.8-fold mRNA; 2.0 ± 0.1-fold protein) and significantly inhibited cell proliferation (72.2 ± 6.9%), migration (62.1 ± 2.8%), tubulogenesis (48.2 ± 4.7%), TRX activity (59.8 + 4.8%) and VEGF (65.6 ± 14.4%) vs. 5 mM control (p < 0.05). TXNIP siRNA rescued these impaired parameters, restoring levels to 96.2 ± 2.1%, 146.2 ± 27.5%, 65.5 ± 13.8%, 73.5 ± 7.9% and 186.6 ± 18.5% of 5 mM control, respectively. DM was associated with a profound
doi:10.1016/j.hlc.2009.05.601 556 HDL INDUCES ANERGY TO PROINFLAMMATORY CYTOKINES IN ENDOTHELIAL CELLS THROUGH A MILD OXIDATIVE STRESS RESPONSE
1 The
Heart Research Institute, Sydney, Australia of Sydney, Sydney, Australia 3 University of Melbourne, Melbourne, Australia 2 University
Background: This study tested whether antiinflammatory effects of HDLs are secondary to their ability to induce a mild oxidative stress response. Methods: Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for up to 16 h. Superoxide (O2 − ) and H2 O2 levels, measured by HE and DCFH-DA respectively, were decreased by treating cells with 50 M butyl hydroxytolune (BHT). Apocynin (250 M), rotenone (1 M) or allopurinol (0.5 mM) was used to block O2 − generation from NAPPH oxidase (NOX), mitochondrial oxidase and xanthine oxidase, respectively. Gene expression was measured by microarray and verified by real-time PCR. Results: Microarray identified 26 oxidative stress genes that were regulated by rHDLs. PCR confirmed significant (p < 0.05) up-regulation of DHCR24 (8-fold), PTGS2 (5-fold), GSR (1.8-fold, PRDX1 (2.3-fold), SOD1 (1.2-fold), TXNRD1 (2-fold), SELS (2.1-fold), NOX5 (1.5-fold) and BNIP3 (1.3-fold) indicating that HDLs initiate a mild oxidative stress response. rHDLs also increased O2 − levels by 20% (p < 0.05) with an associated 1.5–2.0-fold increase in the redox sensitive genes, VCAM-1 and RANTES (p < 0.05). The source of the O2 − was mitochondrial oxidase. HDLs induced the mild oxidative stress response in HCAECs within 1 h of exposure with the antioxidant gene expression apparent by 8 h and maximal at 16 h. Conclusion: rHDLs induce a mild oxidative stress response in HCAECs that results in a small increase in intracellular ROS levels and subsequent induction of a strong antioxidant response. doi:10.1016/j.hlc.2009.05.602
ABSTRACTS
Heart, Lung and Circulation 2009;18S:S1–S286
S243
years. 54% were men, and 15% were current smokers at baseline. The 4-year predicted risk of CHD was overestimated by 198% (95% confidence interval [95% CI]: 162–238%) with UKPDS, and by 146% (117–179%) and 289% (243–341%) using two different Framingham equations. The area under the receiver-operating curves characteristics (95% CI) for the UKPDS equation was 0.692 (0.660–0.723), and 0.650 (0.616–0.684) and 0.666 (0.634–0.699) for the Framingham equations. Within categories of predicted probabilities, the three equations showed a significant lack of fit, with always a systematic overestimation of the risks. The results were consistent for men and women, Caucasians and non-Caucasians. Conclusions: Uncritical application of the Framingham and UKPDS risk function to a contemporary population with diabetes is likely to overestimate coronary risk, which has important implications for cardiovascular preventative strategies. Enhanced risk prediction tools are needed to reliably tailor such strategies in people with diabetes.
impairment of blood flow recovery following hindlimb ischaemia (0.336 ± 0.034 vs. 0.525 ± 0.021; p < 0.0002). However, i.m. injection of Txnip siRNA dramatically increased limb perfusion in diabetic mice to levels nearing control (0.443 ± 0047; p = 0.153). Capillary density was significantly increased by Txnip siRNA (2.7 ± 0.8-fold), while ischaemia (0.0 ± 0.0 vs. 0.5 ± 0.1) and foot movement scores (0.2 ± 0.1 vs. 0.8 ± 0.2) also significantly improved (p < 0.04). Conclusions: The TRX/TXNIP system plays a key role in angiogenesis and impaired neovascularisation in DM. Our findings demonstrate that inhibition of TXNIP can abrogate the deleterious effects of hyperglycaemia on angiogenesis.
doi:10.1016/j.hlc.2009.05.600
A.K. Heather 1,2,3, , X.H. Li 1,2,3 , K.C.Y. McGrath 1,2,3 , K.A. Rye 1,2,3 , P.J. Barter 1,2,3
555 GENE SILENCING OF THIOREDOXIN INTERACTING PROTEIN DRAMATICALLY RESCUES DIABETESRELATED IMPAIRMENT OF ANGIOGENESIS IN VITRO AND IN VIVO L.L. Dunn 1 , A.M. Buckle 1 , P. Simpson 1 , R.W.Y. Chow 1 , D. Sieveking 1 , M.K. Ng 1,2 1 Heart
Research Institute, Sydney, Australia of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia
2 Department
Background: We recently identified a role for thioredoxin (TRX) and its endogenous inhibitor, thioredoxin interacting protein (TXNIP) in angiogenesis. As TXNIP is highly induced by glucose, we hypothesise that the TXNIP/TRX system is dysregulated in diabetes mellitus (DM) leading to impaired angiogenesis. Methods: (i) Human umbilical vein endothelial cells (HUVECs) were cultured in 5–25 mM glucose and siRNA utilised to modulate TXNIP. Angiogenic assays included: cell proliferation, migration, Matrigel tubulogenesis, TRX activity and VEGF ELISA. (ii) Unilateral hindlimb ischaemia was introduced in a murine model of streptozotocin-induced DM. Plasmid encoding Txnip siRNA or scrambled control was injected intramuscularly (i.m.) and limb perfusion assessed by Laser Doppler. Ischaemia, foot movement and capillary density were assessed. Results: Hyperglycaemia (25 mM) induced TXNIP in HUVECs (5.1 ± 1.8-fold mRNA; 2.0 ± 0.1-fold protein) and significantly inhibited cell proliferation (72.2 ± 6.9%), migration (62.1 ± 2.8%), tubulogenesis (48.2 ± 4.7%), TRX activity (59.8 + 4.8%) and VEGF (65.6 ± 14.4%) vs. 5 mM control (p < 0.05). TXNIP siRNA rescued these impaired parameters, restoring levels to 96.2 ± 2.1%, 146.2 ± 27.5%, 65.5 ± 13.8%, 73.5 ± 7.9% and 186.6 ± 18.5% of 5 mM control, respectively. DM was associated with a profound
doi:10.1016/j.hlc.2009.05.601 556 HDL INDUCES ANERGY TO PROINFLAMMATORY CYTOKINES IN ENDOTHELIAL CELLS THROUGH A MILD OXIDATIVE STRESS RESPONSE
1 The
Heart Research Institute, Sydney, Australia of Sydney, Sydney, Australia 3 University of Melbourne, Melbourne, Australia 2 University
Background: This study tested whether antiinflammatory effects of HDLs are secondary to their ability to induce a mild oxidative stress response. Methods: Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for up to 16 h. Superoxide (O2 − ) and H2 O2 levels, measured by HE and DCFH-DA respectively, were decreased by treating cells with 50 M butyl hydroxytolune (BHT). Apocynin (250 M), rotenone (1 M) or allopurinol (0.5 mM) was used to block O2 − generation from NAPPH oxidase (NOX), mitochondrial oxidase and xanthine oxidase, respectively. Gene expression was measured by microarray and verified by real-time PCR. Results: Microarray identified 26 oxidative stress genes that were regulated by rHDLs. PCR confirmed significant (p < 0.05) up-regulation of DHCR24 (8-fold), PTGS2 (5-fold), GSR (1.8-fold, PRDX1 (2.3-fold), SOD1 (1.2-fold), TXNRD1 (2-fold), SELS (2.1-fold), NOX5 (1.5-fold) and BNIP3 (1.3-fold) indicating that HDLs initiate a mild oxidative stress response. rHDLs also increased O2 − levels by 20% (p < 0.05) with an associated 1.5–2.0-fold increase in the redox sensitive genes, VCAM-1 and RANTES (p < 0.05). The source of the O2 − was mitochondrial oxidase. HDLs induced the mild oxidative stress response in HCAECs within 1 h of exposure with the antioxidant gene expression apparent by 8 h and maximal at 16 h. Conclusion: rHDLs induce a mild oxidative stress response in HCAECs that results in a small increase in intracellular ROS levels and subsequent induction of a strong antioxidant response. doi:10.1016/j.hlc.2009.05.602
ABSTRACTS
Heart, Lung and Circulation 2009;18S:S1–S286