Zamaneh Kassiri, PhD Associate Professor Department of Physiology University of Alberta. May 27, 2016

Transcription

1 Zamaneh Kassiri, PhD Associate Professor Department of Physiology University of Alberta May 27, 2016

2

3 Fluorescent microscope PSR staining/confocal microscopy Electron microscope Scanning electron microscopy

4 Cell digestion/removal

5 Fan, Creemers & Kassiri, Circulation Research 2014

6 (Picrosirius Red staining and confocal microscopy) Excess degradation Excess Accumulation (Fibrosis) Modified from Kassiri et al., Circulation Research, 2005

7

8 TIMP TIMP TIMP TIMP TIMP MMPs CONSTRUCTIVE Production, Assembly, and Deposition of ECM proteins Intact MATRIX structure DESTRUCTIVE Degradation/turnover of ECM proteins. Proteolytic Axis

9 Domain Structure of Metalloproteinases Gelatin-binding Simple Hemopexin Furin-activated Domain Minimal Domain Transmembrane GPI-linked Takawale, Kassiri, Comp. Physiol Zhang, Shen, Kassiri, J Mol Cell Cardiol 2015

10 High degree of homology among TIMPs C-terminus N-terminus General structure of a TIMP molecule Disulfide bonds at 12 strictly conserved Cystein residues maintain the six loop structure. Visse & Nagase, Circ Res 2003

11 The N-terminal cysteine in TIMP chelates the active-site zinc in the catalytic domain of MMP. TIMP MMP Catalytic site Visse & Nagase, Circ Res 2003

12 The function and contribution of the ECM, and its regulatory factors, TIMPs, MMPs and ADAMs in heart & vascular diseases. Experimental models of human heart diseases (MI, TAC, agonist-induced hypertrophy/fibrosis). Experimental models of human vascular diseases (hypertension, atherosclerosis, aortic aneurysm:taa, AAA).

13 In 2003, TIMP3 was reported to be anti-angiogenic VEGF-B VEGF-A VEGF-C VEGF-D VEGF-E TIMP3 Extracellular space Intracellular space VEGFR-1VEGFR-2 VEGFR-3 Activation of Macrophages Angiogenesis Angiogenesis in the early Embryo, Lymphanogenesis Question : Would TIMP3-deficiency promote angiogenesis?

14

15 Regional analyses Infarct Peri-infarct WT and TIMP3 -/- LAD Ligation Non-Iinfarct Echocardiography (Vevo 3100) LAD ligation Day 3 inflammation, Proteolytic activities, coronary density (early events) Day 7 Function (echo), infarct expansion, Angiogenesis/coronary density. Day 28 Function (echo), Infarct expansion, Antiogenesis/ coronary density

16 1 mm Survival (%) Cumulative Rupture incidence (%) WT MI in TIMP3 -/- mice increased rate of LV rupture (and mortality), LV dilation, LV systolic and diastolic dysfunction compared to WT-MI. 100 MI Total survival 100 Rupture incidence Post-MI sham 3 days 1 week 4 weeks WT TIMP3 -/ Time (days) TIMP3 -/ MI Time (days) WT TIMP3 -/- WT-Sham WT-MI TIMP3 -/- Sham TIMP3 -/- -MI Aorta Aorta Apex Aorta Aorta Apex Apex Apex Kandalam & Basu et al. AJP: Heart and Circulatory Physiology, 2010.

17 TIMP3 -/- WT Sham Infarct Non-Infarct Second Harmonic Generation (SHG) Imaging (unfixed and unstained tissue) Modified from Kandalam et al., AJP: Heart & Circulatory Phys. 2010

18

19 TIMP3 proteins levels are reduced early (1 day) post-mi TIMP1 Loading control sham infarct Peri-infarct Non-infarct TIMP2 Loading control TIMP3 Loading control TIMP4 Loading control

20 LAD ligation Infarcted myocardium LV chamber Peri-infarct

21 sham Ad-Null Ad-TIMP3 Sham Ad-Null Ad-TIMP3 Sham Ad-Null Ad-TIMP3 Over-expression of TIMP3 improved LV contractility and reduced LV dilation post-mi WT Sham MI +Ad-null MI+Ad-hTIMP3 Apex Aorta Apex Aorta Apex Aorta Improved contractility Reduced dilation WMSI * * * * * * MI MI MI

22 Improved adverse remodeling within the infarcted area in Ad-TIMP3 group Ad-Null WT-MI + Ad-TIMP3 Infarct (40X) Infarct (200X) Peri-infarct (200X)

23 Non-Infarct Peri-Infarct Infarct Ad-TIMP3 delivery results in more viable Myocytes in the infarct region (1 wk post-mi) MI+ Ad-Null Phalloidin WGA MI+ Ad-hTIMP3 25 µm

24 Non-Infarct Peri-Infarct Infarct Higher Vascular Density detected in Ad-TIMP3-injected hearts (FITC-lectin injection) MI+ Ad-Null MI+Ad-hTIMP3 50 µm 25 µm

25 Non-Infarct Peri-Infarct Infarct CD31 Staining (endothelial cells) confirms higher vascular density in Ad-hTIMP3-injected hearts MI+ Ad-Null MI+ Ad-hTIMP3 50 µm 25 µm

26 # of sprouts / bead Length of sprouts The pro-angiogenic effects of TIMP3 is concentration-dependent VEGF A (50 ng/ml) rtimp3 10 ng/ml 100 ng/ml ng/ml No growth factor (-ve control) 80 µm VEGF (50ng/ml) + rtimp VEGF (50ng/ml) + rtimp3

27 IP: TIMP3 IB: VEGFR2 VEGFR2 (210, 230 kd) IP: TIMP3 IB: TIMP3 TIMP3 (25 kd)

28 Replenishing TIMP3 early post-mi can be an effective therapy in post-mi recovery (limits infarct expansion, reduces LV dilation, and improves function). TIMP3 promotes angiogenesis by triggering EC sprouting (in contrast to the conventional dogma that TIMPs are anti-angiogenic). Angiogenic function of TIMP3 is concentration-dependent. TIMP3 has diverse function in the heart (and other organs).

29 Traditional understanding of the function of TIMPs Novel understanding of TIMPs functions

30 Lab members Dr. Xiuhua (Sue) Wang (Lab Technician/surgeon) Lab Alumni Summer Students Post-doctoral Fellows Ji-Won Lee (MSc) ( ) Sid Pamnani Lu Qiao Michael Chute Dr. Dong Fan Dr. Siva Sakamuri Dr. Wang Wang Ratnadeep Basu (MD/PhD) ( ) Aaron Fong PhD students Vijay Kandalam (PhD) ( ) Abhijit Takawale Mengcheng Shen Pu (Pauling) Zhang Ahmed Awad (MSc) ( )

31 UNIVERSITY OF ALBERTA Outside of U of A Allan Murray (angiogenesis) Sandra Davidge (Hypertension) Carlos Fernandez-Patron (ADAMs) Gavin Oudit (Explanted Heart) Richard Lehner (TIMPs & metabolism) Spencer Proctor (TIMPs/atherosclerosis) Darren Freed (fibrosis & matrix) Sean McMurtry (Aneurysm) Gary Lopaschuk Douglas Mann (Washington U in St. Louis) Carl Blobel (Cornell University) Paul Fedak (U of Calgary) Michael Czubryt (U of Manitoba) Diane Jaworski ( U of Vermont) CVRC core: Donna Becker (echocardiography) Amy Barr (Adenovirus amplification)

32 Edmonton, Alberta, Canada

33 The composition of the matrix needs to be considered in drug development I have to go through all that just to get to the cells? Drug

34 Microtubules mediate intracellular exchange between cfb & cmyo (role of ECM in formation of these microtubules?) C He et al. Cardiovascular Research 2011 Fan, Creemer & Kassiri Circ Res 2014

35

36 Electron microscope