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Endothelial Cells: the Vascular Mirror of Metabolic Derangement Loredana Bucciarelli UO di Endocrinologia e Diabetologia Istituto Clinico Humanitas IRCCS.

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Presentation on theme: "Endothelial Cells: the Vascular Mirror of Metabolic Derangement Loredana Bucciarelli UO di Endocrinologia e Diabetologia Istituto Clinico Humanitas IRCCS."— Presentation transcript:

1 Endothelial Cells: the Vascular Mirror of Metabolic Derangement Loredana Bucciarelli UO di Endocrinologia e Diabetologia Istituto Clinico Humanitas IRCCS Ninth International Symposium HEART FAILURE & Co. Rozzano (MI), April 17-18, 2009

2 The Ecs Mirror the Vascular Dysfunction Inflammation and endothelial dysfunction as therapeutic targets in patients with heart failure Dimitris Tousoulis,, Marietta Charakida and Christodoulos Stefanadis Int J of Cardiology Vol 100 Issue

3 Hemostasis Inflammation Vascular toneAngiogenesis and Apoptosis Endothelium Endothelitis

4 Arterial Endothelial Biopsy Feng L et al. Radiology. 1999; 212:655.

5 Venous Endothelial Biopsy 3 J-wires per each vein access were sequentially inserted through a 20- gaugeangiocath pediatric J-wire Endothelial cells were harvested from a forearm vein by scraping

6 Advantages of venous vs. arterial endothelial biopsy Venous endothelial biopsy is less invasive and more relialble Requires little training and technical expertise It is a procedure which can be performed repetitively in patients, even in the out-coming patients. It represent a reliable, minimally invasive approach to monitor the expression of genes in the endothelium over time, and to correlate it with clinical development or progression of vascular disease (i.e. diabetic vascular complications)

7 1) Biopsy 3 wires each arm 3) EC isolation incubation with magnetic Beads coniugated with ab anti CD146 Within 60’ RNA extraction and amplification Endothelial Biopsy microarray Real time PCR 2) Slide preparation for Protein immunofluorescent analysis

8 Protein Analysis Quantitative Immunofluorescence Protein of interest Nucleus (DAPI) Von Willebrand Gene expression analysis Purification RNA extraction Amplification Microarray analysis Real time PCR Microarray Analysis Real time PCR

9 Purification of Endothelial Cells using Magnetic Beads endothelial specific CD146 EC leukocytes iron bead antibody magnet

10 Death Atherosclerosis LVH Risk factors Adapted from Dzau V, Braunwald E. Am Heart J. 1991;121: Sudden death Myocardial infarction Neurohormonal activation Arrhythmia and loss of muscle Remodeling Ventricular dilatation Heart failure Coronary thrombosis Myocardial ischemia CAD Diabetes Hyperlipidemia Hypertension Insulin resistance Chain of Events Leading to CV Mortality

11 Epidemiology of Diabetic Heart Failure Framingham study (risk of HF in diabetics vs nondiabetics) 1 –2X diabetic males –5X diabetic females –4X young diabetic males –8X young diabetic females US HMO prevalence study 2 –With diabetes, HF developed at a rate of 3.3% per year Each 1% elevation in HbA1c leads to a 15% increase in risk of HF 3 1.Kannel WB et al. JAMA. 1979;241: Nichols GA. Diabetologia. 2000;43(suppl A2):7. 3.Chue CU et al. Circulation. 1998;98(suppl 1):721.

12 Hemostasis Inflammation Vascular toneAngiogenesis Apoptosis Endothelium

13 NF-kB-IkBα IkBα NF-kB pro-inflammatory genes COX-2 iNOS ICAM, VCAM E-selectin, P- selectin Tissue factor IL-6, IL-8, RAGE, EGR-1, MCP-1 endothelial cell Cytokines (TNF-α and IL-1ß) Oxidative stress In Inflammatory disease Endothelial activation

14 ROS production antioxidant capacity ROS production antioxidant capacity OXIDATIVE STRESS ! Oxidative balance Superoxide dismutase (SOD) Gluthatione peroxidase (GPX) Catalase

15 NO balance NO production O2-O2- Hypertension Hypercholesterolaemia Diabetes Smoking CAD CHF O2-O2- NO production Endothelial Dysfunction !

16 Linke A, e al. Heart Failure Reviews, 2003;8:87

17 Endothelial dysfunction: therapeutic interventions in heart failure Stimulating NO synthesis Statins L-Arginine Antioxidants ACE inhibitors Tetrahydrobiopterin Estrogens Protecting deactivation of NO Antioxidants Hypolipidemic agents Folic acid/B12/B6, sRAGE Control of diabetes Smoking cessation Diet Exercise

18 Nitrotyrosine COX-2 Artery decompensated CHF Vein decompensated CHF Vein controls Colombo PC, et al. J Appl Physiol. 2002; 92:1331

19 Type 1 Diabetes Murine model C57/blck6 trated with STZ Short term (18wks) and Long term (36 wks) Prove of Purity: RT PCR and IF PCR array: endothelial cell biology plate (84 genes) Confirmation by Taqman Real Time PCR WB of Caspase 3

20 IF: CD31 and Dil-ac-LDL

21 PCR Array in a type 1 Diabetes Murine Model Loredana G. Bucciarelli, Andreas Pollreisz, Anjali Ganda, Moritz Kebschull,Enathia Lalla, Natasha Kalea, Barry Hudson, Ravichandran Ramasamy, Shi Fang, Paolo Colombo and Ann marie Schmidt Inflammatory Stress & Incipient Apoptosis in Primari Venous & Aortic Endothelia Cells of type 1 Diabetic mice. Submitted to Circulation

22 Pentraxin-3: member of the pentraxin superfamily (as CRP), expressed in human atherosclerotic lesions, up-regulated in vitro in ECs by oxLDL PTX-3 Fold change 4.43 parathyroid hormone-like hormone: pro-inflammatory cytokine PTH-LH Fold change Nidogen-2 : Fibrous cap Nid-2 Fold change 7.16 Periodontal Disease Study Selected genes up-regulated in periodontitis patients (n=5) vs. healthy controls (n-4). Gene array expression Protein expression

23 HemostasisInflammation Vascular toneAngiogenesis Endothelium

24 FMD in HF * % Compensated HF Decompensated HF Colombo PC. Circulation. 111:58, 2005

25 FMD in Young Type 1 Diabetics Early Endothelial Dysfunction Twenty-six diabetics (23.4 ± 5.8 years) and 36 healthy volunteers (23.1 ± 2.8 years) were recruited. The duration of diabetes was 9.2 ± 5.3 years; metabolic control was moderate (HbA1c 7.6 ± 1.0%) and IMT was normal in both groups. FMD was significantly impaired in type 1 diabetics (7.13 ± 0.43 vs ± 0.43%; p = 0.002). The FMD grade was associated with diabetes and age. Patients with a good metabolic control (HbA1c ≤ 7.0%) had a better FMD. Conclusion: In type 1 diabetics, even without preclinical or clinical atherosclerosis, endothelial function is already disturbed and can be detected using ultrasonography Hurks R Europ J Vasc Endovasc Surg 2009

26 Endo PAT 2000: Hearing Heart Disease At the Mayo Clinic in Rochester, Minn. 50% percent of pts.who arrive at ER with heart attacks don't have conventional risk factors," such as high cholesterol or blood pressure and…….."Endothelial function may be the missing link." The study's senior author is Mayo cardiologist Dr. Amir Lerman It will become routine at Mayo in the next 2 years the technology, which involves "listening" to minute vascular functions through sensors attached to a patient's index fingers and interpreting the readings via software. Results are presented on a scale from 1 to 5: Healthy adults score around 3, while a mark below 1.7 raises “red flags” The EndoPAT was approved by the U.S. Food and Drug Administration in 2003

27 Endopat 2000

28 Deanfield, J. E. et al. Circulation 2007;115: Oxidative stress induces senescence of endothelial cells. This is reflected in detachment of endothelial cells or parts of the endothelial cell membrane (endothelial microparticles (EMPs)) Vascular integrity becomes dependent on the incorporation of circulating progenitor cells (EPCs) Endothelial Remodeling

29 COMPREHENSIVE ENDOTHELIAL ANALYSIS Bedside to Bench and Back Functional Analysis Remodeling Analysis Molecular Analysis Gene expression Protein expression Endo-PAT 2000 PAT (Peripheral Arterial Tone) Flow-Mediated Dilation Circulating EPC (repair) Circulating EMP (apoptosis)

30 CHF study Type 1 and type 2 diabetes studies ESRD study pre and post dialysis LES study Model of congestive venous flow Obstructive sleep apnea and c-PAP treatment (Jelic s et al Circulation april 2008) Periodontal disease study (submitted) Type 2 diabetes study (Endopat 2000) Ongoing and future studies

31 Future Directions Endothelial biopsy Strict therapeutic control Endothelial biopsy 6 months Comparison patients vs control subjects Evaluation of patients pre and post optimized therapy (longitudinal study) and comparison vs control subjects

32 Thanks to: Istituto Clinico Humanitas Rozzano, MI Prof. Alberto Mantovani Dott. Stefano Genovese Dott. Edoardo Gronda Columbia University New Y ork, NY Prof.ssa Ann marie Schmidt Dott. Ravichandran Ramasamy Dott. Shi Fang Yan Dr. Paolo Colombo Prof. Panos Papapanou

33 Real Time PCR

34 COMPREHENSIVE ENDOTHELIAL ANALYSIS Bedside to Bench FMD conduit arteries Endo-PAT2000 Resistance arteries Circulating EPC Endothelial repair Circulating EMP Endothelial apoptosis eNOS Phospho-eNOS iNOS Catalase GPX COX-2 Egr-1 RAGE Nitrotyrosine Pentraxin 3 Tissue factor PAI-1 Birc2 Naip1 Rhob Casp3 Functional Analysis Remodeling Analysis Molecular Analysis Vasoactive Pro- Inflammatory Oxidant/ anti-oxidant Prothrombotic Flow Cytometry Hand vein LVDT conduit veins Apoptosis

35 Summary We esthablishe a new method to isolate ECs from Aorta and Vena Cava We demonstrated that the veins are the mirror of the arterial site. Genes participating in EC activation included Fibroblast Growth Factor 1 (Fgf 1), Thrombospondin 1 (Thbs1), Chemokine C-C motif ligand 5 (Ccl5), Tissue factor pathway inhibitor (Tfpi), Chemokine C-C motif ligand 2 (Ccl2), Interleukin beta 1 (Ilb1), and Matrix metalloproteinase-2 (Mmp2) and genes participating in EC injury/apoptosis included Receptor (TNFRSF)-interacting serine-threonine kinase 1 (Ripk1), Ras Homolog Gene Family Member B (Rhob), and Caspase 1 were upregulated in the diabetic mice versus the control mice in both aortic and venous ECs.

36 Linear RNA Amplification

37 Nuclear stainingVon Willebrand 10μm μm COX-2COX-2 (digitized and processed) 10μm Quantitative Immunofluorescence Microscopy

38 pediatric J-wire Three J-wires are inserted through an angiocath and used to harvest endothelial cells from a superficial forearm vein by scraping Colombo PC et al. J Appl Physiol. 2002; 92:1331

39 Healthy subjects Compensated CHF Decompensated CHF B) COX * ‡ iNOSD) 2 † § C)eNOS A) Nitrotyrosine * ‡ Colombo PC. Circulation. In press. Colombo PC. Circulation. 111:58, 2005 colombo: Il moglioramento del quadro clinico si accompagnava ad una regressione dell’attivazione endoteliale evidenziando cosi’ una transizione verso la quiete eNOS endoteliale colombo: Il moglioramento del quadro clinico si accompagnava ad una regressione dell’attivazione endoteliale evidenziando cosi’ una transizione verso la quiete eNOS endoteliale Endothelial Activation Quiescence

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