Ninth International Symposium HEART FAILURE Co Rozzano

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

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 2005

Endothelitis Hemostasis Inflammation Endothelium Vascular tone Angiogenesis and Apoptosis

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

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

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)

Endothelial Biopsy 1) Biopsy 3 wires each arm 2) Slide preparation for Protein immunofluorescent analysis 3) EC isolation RNA extraction and amplification microarray Real time PCR Within 60’ incubation with magnetic Beads coniugated with ab anti CD 146

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

Purification of Endothelial Cells using Magnetic Beads CD 146 EC leukocytes endothelial specific iron bead antibody magnet

Chain of Events Leading to CV Mortality Coronary thrombosis Myocardial ischemia Myocardial infarction Arrhythmia and loss of Sudden death Neurohormonal muscle activation Remodeling CAD Ventricular dilatation Atherosclerosis LVH Diabetes Risk factors Hyperlipidemia Hypertension Insulin resistance Adapted from Dzau V, Braunwald E. Am Heart J. 1991; 121: 1244 -1263. Heart failure Death

Hemostasis Inflammation Endothelium Vascular tone Angiogenesis Apoptosis

Cytokines (TNF-α and IL-1ß) Oxidative stress NF-k. B-Ik. Bα NF-k. B Ik. Bα endothelial cell Endothelial activation In Inflammatory disease pro-inflammatory genes COX-2 i. NOS ICAM, VCAM E-selectin, Pselectin Tissue factor IL-6, IL-8, RAGE, EGR-1, MCP-1

ROS production antioxidant capacity Superoxide dismutase (SOD) Gluthatione peroxidase (GPX) Catalase Oxidative balance ROS n ductio ro p OXIDATIVE STRESS ! nt ioxida ant ity capac

O 2 - NO produ ction • Hypertension • Hypercholesterolaemia • Diabetes NO balance • Smoking • CAD • CHF - O 2 NO ion oduct pr Endothelial Dysfunction!

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

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

Type 1 Diabetes Murine model • • C 57/blck 6 trated with STZ Short term (18 wks) 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

IF: CD 31 and Dil-ac-LDL

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

Periodontal Disease Study Protein expression Gene array expression • • Pentraxin-3: member of the pentraxin superfamily (as CRP), expressed in human atherosclerotic lesions, up-regulated in vitro in ECs by ox. LDL PTX-3 Fold change 4. 43 • parathyroid hormone-like hormone: pro -inflammatory cytokine PTH-LH Fold change 10. 72 • Nidogen-2 : Fibrous cap Nid-2 Fold change 7. 16 Selected genes up-regulated in periodontitis patients (n=5) vs. healthy controls (n-4).

Inflammation Hemostasis Endothelium Vascular tone Angiogenesis

FMD in HF 12 * 10 % 8 6 4 2 0 Decompensated HF Colombo PC. Circulation. 111: 58, 2005

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 (Hb. A 1 c 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 (Hb. A 1 c ≤ 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

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 Endo. PAT was approved by the U. S. Food and Drug Administration in 2003

Endopat 2000

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: 1285 -1295

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

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)

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

Thanks to: Istituto Clinico Humanitas 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

Real Time PCR

COMPREHENSIVE ENDOTHELIAL ANALYSIS Bedside to Bench Molecular Analysis Vasoactive Oxidant/ anti-oxidant Pro. Inflammatory Prothrombotic Apoptosis e. NOS Phospho-e. NOS i. NOS Catalase GPX COX-2 Egr-1 RAGE Nitrotyrosine Pentraxin 3 Tissue factor PAI-1 Birc 2 Naip 1 Rhob Casp 3 Functional Analysis FMD conduit arteries Endo-PAT 2000 Resistance arteries Remodeling Analysis Circulating EPC Endothelial repair Circulating EMP Endothelial apoptosis Hand vein LVDT conduit veins Flow Cytometry

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 (Thbs 1), Chemokine C-C motif ligand 5 (Ccl 5), Tissue factor pathway inhibitor (Tfpi), Chemokine C-C motif ligand 2 (Ccl 2), Interleukin beta 1 (Ilb 1), and Matrix metalloproteinase-2 (Mmp 2) and genes participating in EC injury/apoptosis included Receptor (TNFRSF)-interacting serine-threonine kinase 1 (Ripk 1), 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.

Linear RNA Amplification

Quantitative Immunofluorescence Microscopy 10 μm Nuclear staining 10 μm Von Willebrand 10 μm COX-2 (digitized and processed)

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

B) COX-2 A) Nitrotyrosine 5 ‡ 4 * 3 ‡ 8 6 4 2 * 2 1 0 0 1 2 3 C) e. NOS D) i. NOS 1 3 3 2 1. 5 2 § † 1 1 1 0. 5 0 0 1 2 3 Decompensated CHF Compensated CHF Healthy subjects Endothelial Activation Quiescence Colombo PC. Circulation. In press. Colombo PC. Circulation. 111: 58, 2005