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Endothelial Cells: the Vascular Mirror of Metabolic Derangement Loredana Bucciarelli

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.

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Endothelial Cells: the Vascular Mirror of Metabolic Derangement Loredana Bucciarelli

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  1. 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 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

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

  8. Gene expression analysis • Purification • RNA extraction • Amplification • Microarray analysis • Real time PCR Protein AnalysisQuantitative 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 Myocardialinfarction Coronarythrombosis Sudden death Arrhythmia and loss of muscle Neurohormonalactivation Myocardialischemia Remodeling CAD Ventriculardilatation AtherosclerosisLVH Heartfailure Diabetes Hyperlipidemia Hypertension Insulin resistance Risk factors Death Adapted from Dzau V, Braunwald E. Am Heart J. 1991;121:1244-1263.

  11. Hemostasis Inflammation Endothelium Vascular tone Angiogenesis Apoptosis

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

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

  14. O2- NO production • Hypertension • Hypercholesterolaemia • Diabetes • Smoking • CAD • CHF NO balance NO production O2- Endothelial Dysfunction!

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

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

  17. 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

  18. IF: CD31 and Dil-ac-LDL

  19. 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

  20. Periodontal Disease Study Protein expression Gene array 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-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

  21. Inflammation Hemostasis Endothelium Vascular tone Angiogenesis

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

  23. 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

  24. 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

  25. Endopat 2000

  26. 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

  27. COMPREHENSIVE ENDOTHELIAL ANALYSISBedside to Bench and Back 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)

  28. 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)

  29. Future Directions • Comparison patients vs control subjects • Evaluationof patients pre and post optimized therapy (longitudinal study) and comparison vs control subjects Endothelial biopsy Endothelial biopsy Strict therapeutic control 6 months

  30. 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

  31. Real Time PCR

  32. Molecular Analysis Functional Analysis Remodeling Analysis COMPREHENSIVE ENDOTHELIAL ANALYSIS Bedside to Bench 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

  33. 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.

  34. Linear RNA Amplification

  35. Quantitative Immunofluorescence Microscopy 10 μ m Nuclear staining Von Willebrand 10 10 μ μ m m COX-2 COX-2 (digitized and processed)

  36. 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

  37. B) COX-2 A) Nitrotyrosine ‡ ‡ 5 8 4 * 6 3 4 * 2 2 1 0 0 3 1 2 Endothelial Activation Quiescence C) eNOS D) iNOS 1 3 3 2 § 1.5 † 2 1 1 1 0.5 0 0 1 2 3 colombo: Il moglioramento del quadro clinico si accompagnava ad una regressione dell’attivazione endoteliale evidenziando cosi’ una transizione verso la quiete eNOS endoteliale Healthy subjects Compensated CHF Decompensated CHF Colombo PC. Circulation. In press. Colombo PC. Circulation. 111:58, 2005

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