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OXIDIZED LOW-DENSITY LIPOPROTEINS REDUCE MICRO-RNA 146B EXPRESSION AND LEAD TO ENDOTHELIAL DYSFUNCTION: ROLE OF TRAF6 SIGNALING PATHWAY
Abstracts
334 OXIDIZED LOW-DENSITY LIPOPROTEINS REDUCE MICRO-RNA 146B EXPRESSION AND LEAD TO ENDOTHELIAL DYSFUNCTION: ROLE OF TRAF6 SIGNALING PATHWAY M Desjarlais, S Dussault, V Sanchez, S Harel, S Hussain, A Rivard Montréal, Québec BACKGROUND:
Oxidized low-density lipoproteins (oxLDLs) contribute to the dysregulation of endothelial cell functions, an initial key step involved in atherosclerosis. Endothelial dysfunction is characterized by a pro-inflammatory state, adhesion of circulating monocytes, and impairment of angiogenesis. Micro RNAs (miRNAs) are small noncoding RNAs that are emerging as key regulators of several physiological and pathological processes. However, their precise role in the development of endothelial dysfunction and atherosclerosis remains to be determined. Because miR-146b was previously documented to be an important inhibitor of inflammation, we investigated its potential role in oxLDLinduced endothelial dysfunction. METHODS AND RESULTS: The effect of oxLDL exposure on the expression of miR-146b was studied in Human Umbilical Vein Endothelial Cells (HUVECs). qRT-PCR analyses showed that miR-146b levels are reduced by more than 90% in HUVECs treated with oxLDLs for 24 hours. Exposure of HUVECs to oxLDLs is also associated with higher oxidative stress levels, increased monocyte (THP-1) adhesion, decreased cellular migration, and impaired tubule formation. To better characterize the role of miR-146b, HUVECs were transfected with an inhibitor of miR-146b (AntagomiR-146b) or a control AntagomiR. Similarly to oxLDL exposure, we found that inhibition of miR-146b in HUVECs leads to increased oxidative stress and THP-1 adhesion, together with an impairment of angiogenic functions (migration and tubule formation). miR-146b has previously been shown to target TNF receptor associated factor 6 (TRAF6), an important regulator of inflammation. At the molecular level, we found that TRAF6 expression is significantly increased in HUVECs exposed to oxLDLs or transfected with antagomiR-146b. In addition, the expression of downstream signaling molecules such as TNF alpha and VCAM-1 is significantly increased after treatment with oxLDL or antagomiR-146b. To confirm the important role of miR-146b in oxLDL-induced endothelial dysfunction, we rescued its expression using a miR mimic. We found that forced expression of miR-146b in HUVECs leads to normalization of TRAF6 inflammatory signaling pathway and protects against the negative effects of oxLDLs including THP-1 adhesion, induction of oxidative stress and impairment of HUVEC angiogenic activities. CONCLUSIONS: oxLDLs reduce miR-146b expression, which leads to endothelial dysfunction. The mechanism involves increased activation of TRAF6 inflammatory pathway. Forced expression of miR-146b using a miR mimic could constitute a
S259
novel therapeutic strategy to maintain endothelial cell functions in atherosclerotic conditions. 335 GEOMETRIC AND MORPHOLOGICAL DETERMINANTS OF ATHEROSCLEROSIS IN THE CAROTID ARTERY S Gregg, T Li, M Hetu, S Pang, A Johri Kingston, Ontario BACKGROUND:
The carotid bifurcation is a common site of atherosclerotic plaque. Plaque development is thought to occur preferentially at geometrically predisposed areas such as arterial branch points. The aim of this study was to investigate the geometric and anatomical variables that contribute to the development of plaque using three-dimensional (3D) ultrasound technology. METHODS: Seventy patients recruited from patients referred for elective coronary angiography underwent 3D carotid ultrasound scans for the purpose of plaque and geometric quantification of the left and right carotid bulbs. Geometric values such as angle, area and length of the carotid bulb and the bifurcation were determined using QLAB software (Philips, Figure 1). Bulb length was measured from the bulb inflow area (BIA) to the bulb outflow cross sectional area (BOCSA) and bulb volume was calculated using the stacked contouring tool, which incorporates bulb length and multiple cross sectional areas. Vessel angles were measured using an electronic protractor tool. For example, the bifurcation angle (BifA) was measured as the angle between the projections of the internal carotid artery (ICA) and the external carotid artery (ECA) vectors. 3D plaque volume within the carotid bulb and artery branches was quantified using the stacked contour method. Relationships between plaque volume and various geometric factors were tested via linear regression. A logistic regression analysis was used to determine the relationship between all variables. RESULTS: Of the 70 patients recruited for this study, 52 out of 70 were men. None of the patients had previously known carotid disease or stroke. The mean age for the total patient population was 65.9 11.5 years. Geometric variables such as bulb inflow area (BIA), bulb volume (BV), bifurcation angle (BifA), internal carotid artery angle (ICAA) and flare were significantly associated with plaque volume. Bulb inflow area (r¼0.28, p¼0.001), BV (r¼0.21, p¼0.01) and BifA (r¼0.18, p¼0.04) showed a positive linear relationship with plaque volume. In contrast, ICAA (r¼-0.18, p¼0.04) and bulb flare (r¼-0.21, p¼0.01) displayed a negative linear relationship with plaque volume. When comparing left and right arteries within individuals, no correlation was found between side and plaque volume. CONCLUSION: Geometric variables were discovered to be potential risk factors associated with plaque volume in the carotid bulb. The BIA had the strongest correlation with plaque volume. Therefore, measurement of the BIA along with BV,
334 OXIDIZED LOW-DENSITY LIPOPROTEINS REDUCE MICRO-RNA 146B EXPRESSION AND LEAD TO ENDOTHELIAL DYSFUNCTION: ROLE OF TRAF6 SIGNALING PATHWAY M Desjarlais, S Dussault, V Sanchez, S Harel, S Hussain, A Rivard Montréal, Québec BACKGROUND:
Oxidized low-density lipoproteins (oxLDLs) contribute to the dysregulation of endothelial cell functions, an initial key step involved in atherosclerosis. Endothelial dysfunction is characterized by a pro-inflammatory state, adhesion of circulating monocytes, and impairment of angiogenesis. Micro RNAs (miRNAs) are small noncoding RNAs that are emerging as key regulators of several physiological and pathological processes. However, their precise role in the development of endothelial dysfunction and atherosclerosis remains to be determined. Because miR-146b was previously documented to be an important inhibitor of inflammation, we investigated its potential role in oxLDLinduced endothelial dysfunction. METHODS AND RESULTS: The effect of oxLDL exposure on the expression of miR-146b was studied in Human Umbilical Vein Endothelial Cells (HUVECs). qRT-PCR analyses showed that miR-146b levels are reduced by more than 90% in HUVECs treated with oxLDLs for 24 hours. Exposure of HUVECs to oxLDLs is also associated with higher oxidative stress levels, increased monocyte (THP-1) adhesion, decreased cellular migration, and impaired tubule formation. To better characterize the role of miR-146b, HUVECs were transfected with an inhibitor of miR-146b (AntagomiR-146b) or a control AntagomiR. Similarly to oxLDL exposure, we found that inhibition of miR-146b in HUVECs leads to increased oxidative stress and THP-1 adhesion, together with an impairment of angiogenic functions (migration and tubule formation). miR-146b has previously been shown to target TNF receptor associated factor 6 (TRAF6), an important regulator of inflammation. At the molecular level, we found that TRAF6 expression is significantly increased in HUVECs exposed to oxLDLs or transfected with antagomiR-146b. In addition, the expression of downstream signaling molecules such as TNF alpha and VCAM-1 is significantly increased after treatment with oxLDL or antagomiR-146b. To confirm the important role of miR-146b in oxLDL-induced endothelial dysfunction, we rescued its expression using a miR mimic. We found that forced expression of miR-146b in HUVECs leads to normalization of TRAF6 inflammatory signaling pathway and protects against the negative effects of oxLDLs including THP-1 adhesion, induction of oxidative stress and impairment of HUVEC angiogenic activities. CONCLUSIONS: oxLDLs reduce miR-146b expression, which leads to endothelial dysfunction. The mechanism involves increased activation of TRAF6 inflammatory pathway. Forced expression of miR-146b using a miR mimic could constitute a
S259
novel therapeutic strategy to maintain endothelial cell functions in atherosclerotic conditions. 335 GEOMETRIC AND MORPHOLOGICAL DETERMINANTS OF ATHEROSCLEROSIS IN THE CAROTID ARTERY S Gregg, T Li, M Hetu, S Pang, A Johri Kingston, Ontario BACKGROUND:
The carotid bifurcation is a common site of atherosclerotic plaque. Plaque development is thought to occur preferentially at geometrically predisposed areas such as arterial branch points. The aim of this study was to investigate the geometric and anatomical variables that contribute to the development of plaque using three-dimensional (3D) ultrasound technology. METHODS: Seventy patients recruited from patients referred for elective coronary angiography underwent 3D carotid ultrasound scans for the purpose of plaque and geometric quantification of the left and right carotid bulbs. Geometric values such as angle, area and length of the carotid bulb and the bifurcation were determined using QLAB software (Philips, Figure 1). Bulb length was measured from the bulb inflow area (BIA) to the bulb outflow cross sectional area (BOCSA) and bulb volume was calculated using the stacked contouring tool, which incorporates bulb length and multiple cross sectional areas. Vessel angles were measured using an electronic protractor tool. For example, the bifurcation angle (BifA) was measured as the angle between the projections of the internal carotid artery (ICA) and the external carotid artery (ECA) vectors. 3D plaque volume within the carotid bulb and artery branches was quantified using the stacked contour method. Relationships between plaque volume and various geometric factors were tested via linear regression. A logistic regression analysis was used to determine the relationship between all variables. RESULTS: Of the 70 patients recruited for this study, 52 out of 70 were men. None of the patients had previously known carotid disease or stroke. The mean age for the total patient population was 65.9 11.5 years. Geometric variables such as bulb inflow area (BIA), bulb volume (BV), bifurcation angle (BifA), internal carotid artery angle (ICAA) and flare were significantly associated with plaque volume. Bulb inflow area (r¼0.28, p¼0.001), BV (r¼0.21, p¼0.01) and BifA (r¼0.18, p¼0.04) showed a positive linear relationship with plaque volume. In contrast, ICAA (r¼-0.18, p¼0.04) and bulb flare (r¼-0.21, p¼0.01) displayed a negative linear relationship with plaque volume. When comparing left and right arteries within individuals, no correlation was found between side and plaque volume. CONCLUSION: Geometric variables were discovered to be potential risk factors associated with plaque volume in the carotid bulb. The BIA had the strongest correlation with plaque volume. Therefore, measurement of the BIA along with BV,