By: Dr Mehrnoosh Shanaki

Transcription

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2 Resveratrol could partly improve the crosstalk between canonical β-catenin/wnt and FOXO pathways in coronary artery disease patients with metabolic syndrome: A case control study By: Dr Mehrnoosh Shanaki Assistant Professor Department of Laboratory Medicine Shahid Beheshti University of Medical Sciences

3 1.Introduction 1

4 Oxidative Stress. MetS, and CAD Cardiovascular events risk factors )Metabolic syndrome component( Insulin resistance Hypertention Obesity Dyslipidemia Oxidative stress Endothelial dysfunction Atherosclerosis MetS: Metabolic Syndrome CAD: Coronary Artery Disease CAD 2

5 FOXO & Wnt signaling pathways MnSOD ROS: Reactive Oxygen species AOX: Antioxidant 3

6 Canonical Wnt/β-Catenin Signaling Pathway Overview of canonical Wnt/β-Catenin Signaling βcell mass/insulin Glucose handling LDL cholesterol Atherosclerosis Blood pressure Bone mass PPAR-δ Ob:Osteoblast Oc:Osteoclast 4

7 Crosstalk between Wnt & FOXO Signaling Pathways under Oxidative Stress Condition MnSOD PPAR-δ Manolagas et al, 2007 Hoogeboom et al, 2008 Shin et al, 2006 Funato et al,

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9 Resveratrol (RES) Resveratrol (3,4,5-trihydroxystilbene): polyphenol compound. 7

10 Cardioprotection effects of RES Atheroprotection Atherosclerosis 8

11 RES Effects on the Crosstalk between Wnt & FOXO Signaling Pathways RES? 9

12 Hypothesis CAD Patients? β-catenin? Does the crosstalk between these two pathways have a role in pathogenesis of CAD (which The Crosstalk itself is between associated Canonical with decrease Wnt and of FOXO pathways antioxidant in CAD defense)? patients has not been studied yet MnSOD PPAR-δ 10

13 Hypothesis? RES? β-catenin? In the present study, we investigate the expression of β-catenin and target-genes of these two pathways in basal condition & after treatment by RES; in order to elucidate the alterations of the crosstalk between these two pathways under this treatment MnSOD PPAR-δ 11

14 2.Materials & Methods 12

15 Experimental Flow chart Study Population Blood Sampling PBMCs Isolation MTT Assay Plasma Separation FRAP Assay Cell Culture RNA Extraction MnSOD Enzyme Activity Total β-catenin protein (EIA) cdna Synthesis Real-time PCR 13

16 Study Population: Laboratory case-control Patient selection Healthy subjects Exclusion criteria Laboratory & anthropometric data recorded 14

17 Patient selection Healthy subjects Men with years Angiographically tested CABG candidates 10 men with MetS and CAD with more than 50% stenosis in at least one coronary vessels. (three vessels CAD patients were selected). 10 Men with years Without MetS MetS criteria is based on NCEP ATP ΙΙΙ (3 0f 5 factors): FBS 100mg/dl Waist circumference 102 cm BP 130/85 mmhg TG 150 mg/dl HDL-C 40 mg/dl CABG: Coronary Artery Bypass Graft ECG: Electrocardiogram 15

18 Exclusion criteria Diabetes Endocrine disorders History of MI or cardiac surgery Severe liver or renal disease Inflammation and Infectious disease Cancers Allergies Smoking Antioxidant supplement consumption Long term / high dose Aspirin Lithium content drugs 16

19 Laboratory & anthropometric data recorded Age Height Weight BMI FBS Lipid profile(tg,tc,ldl-c,hdl-c) Consumed drugs 17

20 PBMCs isolation using Ficoll-Hypaque To Dilute the blood with an equal volume of PBS To layer the diluted blood by Ficoll (1/2 volume of diluted blood) To centrifuge for 30 minutes at 800 g at room temperature To remove carefully the cells from the interface and transfer to a new centrifuge tube. To dilute the transferred cells with PBS to reduce the density of the solution To centrifuge at 400g for 10 minutes to pellet the PBMCs (two times) To resuspend in complete medium containing RPMI1640, 10% FBS,1% pen/strep To assess cell count & PBMC viability with trypan blue Buffy Coat 18

21 MTT assay for cell viability in presence of RES To seed 5 x 10 5 cells/200 µl in 96 well plate To add 200 µl of MTT reagent at final concentration 0.5 mg/ml To incubate at 37 C with 5%CO2 incubator for 24h To incubate at 37 C with 5% CO2 incubator for 4h To remove culture media using centrifugation To remove culture media using centrifugation To add RES(50 &100 µm) for 12 & 24h To add 100 µl of detergent reagent (DMSO) to all wells To remove culture media using centrifugation To leave covered plate in the dark at room temperature for 30 min To wash with PBS 19 To measure the absorbance of the wells, including the blanks, at 540 nm.

22 Cell culture Healthy subjects CAD patients Cont DMSO RES Cont DMSO RES PBMCs in 12 well plate Cell treatments: Control: DMSO(0.025%),12h RES: 50µM,12h In both control and treated cells, DMSO was present at equal concentration (0.025%). 20

23 Cell harvest ( cells) RNA extraction RNA quantitation Spectrophotometery RNA quantification Agarose gel Reverse transcription for cdna synthesis 21 Real time-pcr 2 CT 2 ( CT CT control ) target gene internal

24 Protein & enzyme activity measurement MnSOD enzyme activity assay Total β-catenin protein measurement 22

25 3.Results 23

26 Demographic and clinical data Healthy Subjects N=10 CAD Patients N=10 p-value Characteristics Median ( (range) IQR) Median (IQR) (range) Mann-Whitney test Age (years) 50 (49-51) 51 (50-54) 0.12 Body Mass Index (Kg/m 2 ) 25.1 ( ) ( ) Waist Circumference (cm) 78.5 (72-81) 80.5 (73-86) Total Cholesterol (mg/dl) 173 ( ) 188 ( ) HDL Cholesterol (mg/dl) 40.5 (36-52) 33 (25-41) LDL Cholesterol (mg/dl) 92 (90-95) 98 (92-110) 0.04 Triglyceride (mg/dl) 136 ( ) 166 ( ) Systolic Blood Pressure (mmhg) Diastolic Blood Pressure (mmhg) 120 ( ) 140 ( ) < (80-80) 90 (85-90) <0.001 Fasting Blood Sugar (mg/dl) 88.5 (82-94) 93.5 ( ) 0.03 HDL, High density lipoprotein; LDL, Low density lipoprotein Data are expressed as Median ( Interquartile range )(IQR) Data are expressed as median (range) 24

27 FRAP assay Total plasma antioxidant capacity of CAD patients was significantly 13% (p=0.02) lower than healthy subjects. Data are expressed as means ± SEM, obtained by duplicates of tests. 25

28 MTT assay: PBMCs viability in RES treatments Cells were incubated with 50 and 100 µm RES for 12h and 24h, and cell viability was determined by MTT assay. All RES treatments did not reduce the cell viability less than 90%. Maximal DMSO concentration was 0.025% for all untreated or treated cells. Data are expressed as means ± SEM, obtained by triplicates of tests. * p<0.05; # p<

29 Quantitative Real-time PCR 27

30 Within Group Comparison: Effects of RES on β-catenin mrna expression Healthy Subjects CAD patients Data are expressed as means ± SEM, obtained by duplicates of tests 28

31 Within Group Comparison: Effects of RES on MnSOD mrna expression Healthy Subjects CAD patients Data are expressed as means ± SEM, obtained by duplicates of tests * p<0.05; # p<

32 Within Group Comparison: Effects of RES on PPAR-δ mrna expression Healthy Subjects CAD patients Data are expressed as means ± SEM, obtained by duplicates of tests 30

33 Between Group Comparison: Effects of RES on investigated genes * * Data are expressed as means ± SEM, obtained by duplicates of tests * p<

34 Total β-catenin protein Effects of RES treatment Data are expressed as means ± SEM, obtained by duplicates of tests * p<

35 MnSOD enzyme activity Effects of RES treatment Data are expressed as means ± SEM, obtained by duplicates of tests * p<0.05 Within group comparison (relative to basal condition) p<0.01; Between group comparison 33

36 Pearson correlations Canonical Wnt and FOXO pathways: basal condition mrna β-catenin mrna r = p = r = p = 0.16 r = p < r = p < MnSOD mrna PPAR-δ mrna Protein Total β-catenin protein FOXO r = p = r = p = 0.91 MnSOD Enzyme Activity 34

37 Pearson correlations Canonical Wnt and FOXO pathways: RES treatment mrna β-catenin mrna r = p < r = p = 0.08 r = p = r = p = MnSOD mrna PPAR-δ mrna Protein Total β-catenin protein FOXO r = p = r = p = MnSOD Enzyme Activity 35

38 4.Discussion 36

39 Discussion Plasma total antioxidant capacity (ptac) Basal Condition RES Crosstalk β-catenin MnSOD PPAR-δ Crosstalk β-catenin MnSOD PPAR-δ 37

40 Plasma total antioxidant capacity (ptac) We found ptac of CAD patients was significantly lower than healthy subjects. Several studies were in line with our finding Vassale et al, 2004 The more the number of involved coronary arteries, the less the ptac Shaikh et al, 2009 Nojiri et al, 2001 Khatibi et al, 2013 Park et al, 2009 Demirbag et al, 2010 TAC of plasma and coronary arterial tissues of CAD patients 38

41 Basal Condition crosstalk between canonical Wnt and FOXO pathways Healthy Subjects CAD Patients Low levels of ROS JNK 39

42 RES treatment crosstalk between canonical Wnt and FOXO pathways Healthy Subjects CAD Patients RES RES 40

43 RES effects through β-catenin/foxo pathway: Totalβ-catenin protein & MnSOD activity: Healthy subjects RES F u k u i e t a l, Z h o u e t a l, P I 3 K E R K A k t G S K - 3 b b - C a t e n i n b - C a t e n i n F o x O 41

44 RES effects through alternative pathways: MnSOD activity: CAD patients Robb et al; 2008 Borra et al, 2008 RES Rubiolo et al; 2005 Nrf2: Nuclear factor erythroid 2 related factor 2 42

45 Healthy Subjects CAD Patients RES treatment canonical Wnt RES RES 43

46 Conclusion We observed that FOXO pathway was disrupted in CAD patients and its crosstalk with canonical Wnt was disorganized. It seems CAD patients PBMCs has preferred to employ their limited resource of β-catenin to maintain functions of canonical Wnt, and probably exerted its improvement on antioxidant defense by increasing the expression and activity of MnSOD through other defensive signaling pathways. RES could significantly increase MnSOD enzyme activity independent from β-catenin/foxo. Despite mild improve in β-catenin/foxo pathway under RES treatment, it could not reignite this corrupted signaling pathway. 44

47 Suggestions Altogether, the therapeutic importance of RES in CAD has to be explored by more studies with more extended selected genes and pathways. To assess direct antioxidant effects of RES 45

48 Thank you