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the Vascular Mirror of Metabolic Derangement

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

2 The Ecs Mirror the Vascular Dysfunction
vascular endothelium plays key role in the regulation of vascular tone, in the process of inflammation and in the thrombotic mechanisms 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 Endothelitis Endothelium Inflammation Hemostasis Vascular tone
Angiogenesis and Apoptosis

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

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

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

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

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 study2 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 HF3 Kannel WB et al. JAMA. 1979;241: Nichols GA. Diabetologia. 2000;43(suppl A2):7. Chue CU et al. Circulation. 1998;98(suppl 1):721.

12 Endothelium Hemostasis Inflammation Vascular tone Angiogenesis
Apoptosis Endothelium

13 Cytokines (TNF-α and IL-1ß)
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 OXIDATIVE STRESS ! Oxidative balance ROS antioxidant production
capacity OXIDATIVE STRESS ! Oxidative balance Superoxide dismutase (SOD) Gluthatione peroxidase (GPX) Catalase

15 Endothelial Dysfunction!
NO balance NO production O2- Hypertension Hypercholesterolaemia Diabetes Smoking CAD CHF 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 Artery decompensated CHF Vein controls
Nitrotyrosine COX-2 Artery decompensated CHF Vein controls Vein decompensated CHF 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 Periodontal Disease Study
Gene array expression Protein expression Selected genes up-regulated in periodontitis patients (n=5) vs. healthy controls (n-4). Pentraxin-3: member of the pentraxin superfamily (as CRP), expressed in human atherosclerotic lesions, up-regulated in vitro in ECs by oxLDL  PTX Fold change 4.43 parathyroid hormone-like hormone: pro-inflammatory cytokine PTH-LH Fold change 10.72 Nidogen-2 : Fibrous cap Nid Fold change 7.16

23 Hemostasis Inflammation Vascular tone Angiogenesis Endothelium

24 FMD in HF * % Decompensated HF Compensated HF
12 10 8 6 4 2 % 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. 8.77 ± 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 Endothelial Remodeling
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) Deanfield, J. E. et al. Circulation 2007;115: 28

Molecular Analysis Functional Analysis Remodeling Analysis Gene expression Protein expression Endo-PAT 2000 PAT (Peripheral Arterial Tone) Flow-Mediated Dilation Circulating EPC (repair) Circulating EMP (apoptosis)

30 Ongoing and future studies
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)

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

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

33 Real Time PCR

Bedside to Bench Functional Analysis Remodeling Molecular Vasoactive eNOS Phospho-eNOS iNOS Catalase GPX COX-2 Egr-1 RAGE Nitrotyrosine Pentraxin 3 Tissue factor PAI-1 Birc2 Naip1 Rhob Casp3 Oxidant/ anti-oxidant FMD conduit arteries Circulating EPC Endothelial repair Pro- Inflammatory Endo-PAT2000 Resistance arteries Circulating EMP Endothelial apoptosis Hand vein LVDT conduit veins Prothrombotic Flow Cytometry 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 Quantitative Immunofluorescence Microscopy
Nuclear staining Von Willebrand 10 μ m COX-2 COX-2 (digitized and processed)

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 Endothelial Activation Quiescence
B) COX-2 2 4 6 8 * A) Nitrotyrosine 1 2 3 4 5 * colombo: Il moglioramento del quadro clinico si accompagnava ad una regressione dell’attivazione endoteliale evidenziando cosi’ una transizione verso la quiete eNOS endoteliale C) eNOS 0.5 1 1.5 2 3 3 1 iNOS D) 2 Decompensated CHF Compensated CHF Healthy subjects Endothelial Activation Quiescence Colombo PC. Circulation. In press. Colombo PC. Circulation. 111:58, 2005

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