Endothelial Cells:the Vascular Mirror of Metabolic Derangement

Loredana Bucciarelli

UO di Endocrinologia e Diabetologia

Istituto Clinico Humanitas IRCCS

Ninth International SymposiumHEART FAILURE & Co.Rozzano (MI), April 17-18, 2009

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

HemostasisInflammation

Vascular tone Angiogenesisand Apoptosis

Endothelium

Endothelitis

Arterial Endothelial Biopsy

Feng L et al. Radiology. 1999; 212:655.

Venous Endothelial Biopsy

3 J-wires per each vein access were sequentially inserted through a 20-

gauge angiocathpediatric J-wire

Endothelial cells were harvested from a forearm veinby scraping

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)

1) Biopsy 3 wires each arm

3) EC isolation incubation with magnetic Beads coniugated with ab anti CD146Within 60’

RNA extractionand amplification

Endothelial Biopsy

microarray

Real time PCR

2) Slide preparation for Protein immunofluorescent analysis

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

Purification of  Endothelial Cells using Magnetic Beads

endothelial specific

CD146

ECleukocytes

iron beadantibody

magnet

Death

AtherosclerosisLVH

Risk factors

Adapted from Dzau V, Braunwald E. Am Heart J. 1991;121:1244-1263.

Suddendeath

Myocardialinfarction

Neurohormonalactivation

Arrhythmia and loss of muscle

Remodeling

Ventriculardilatation

Heartfailure

Coronarythrombosis

Myocardialischemia

CAD

DiabetesHyperlipidemiaHypertensionInsulin resistance

Chain of Events Leading to CV Mortality

HemostasisInflammation

Vascular tone AngiogenesisApoptosis

Endothelium

NF-kB-IkBα

IkBα

NF-kB

pro-inflammatory genes

COX-2iNOSICAM, VCAME-selectin, P-selectin Tissue factorIL-6, IL-8, RAGE, EGR-1, MCP-1

endothelial cell

Cytokines (TNF-α and IL-1ß) Oxidative stress

In Inflammatory disease

Endothelial activation

ROSproduction

antioxidantcapacity

ROS

productionantioxidant

capacity

OXIDATIVE STRESS !

Oxidative balance

Superoxide dismutase (SOD)Gluthatione peroxidase (GPX)Catalase

NO balance

NOproduction

O2

-

•Hypertension

•Hypercholesterolaemia

•Diabetes

•Smoking

•CAD

•CHF

O2-

NO

production

Endothelial Dysfunction!

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

0

500

1000

1500

2000

2500

3000

1 2 3

Nitrotyrosine

01000200030004000500060007000

1 2 3

COX-2

Artery decompensated CHF

Vein decompensated CHF

Vein controls

Colombo PC, et al. J Appl Physiol. 2002; 92:1331

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

IF: CD31 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 SchmidtInflammatory Stress & Incipient Apoptosis in Primari Venous & Aortic Endothelia Cells of type 1 Diabetic mice. Submitted to Circulation

•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

Periodontal Disease Study

• Selected genes up-regulated in periodontitis patients (n=5) vs. healthy controls (n-4).

Gene array expression Protein expression

HemostasisInflammation

Vascular tone Angiogenesis

Endothelium

FMD in HF

* 12

10

8

6

4

2

0

%

Compensated HF

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 (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

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

Endopat 2000

Deanfield, J. E. et al. Circulation 2007;115:1285-1295

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

COMPREHENSIVE ENDOTHELIAL ANALYSIS

Bedside to Bench and Back

FunctionalAnalysis

RemodelingAnalysis

MolecularAnalysis

•Gene expression•Protein expression

•Endo-PAT 2000PAT (Peripheral Arterial Tone)

•Flow-Mediated Dilation

•Circulating EPC (repair)•Circulating EMP (apoptosis)

• 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

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

Thanks to:

Istituto Clinico HumanitasRozzano, MI Prof. Alberto MantovaniDott. Stefano GenoveseDott. Edoardo Gronda

Columbia UniversityNew York, NY

Prof.ssa Ann marie SchmidtDott. Ravichandran RamasamyDott. Shi Fang YanDr. Paolo ColomboProf. Panos Papapanou

Real Time PCR

COMPREHENSIVE ENDOTHELIAL ANALYSIS

Bedside to Bench

FMDconduit arteries

Endo-PAT2000Resistance arteries

CirculatingEPC

Endothelial repair

CirculatingEMP

Endothelial apoptosis

eNOSPhospho-eNOSiNOSCatalaseGPXCOX-2Egr-1RAGE NitrotyrosinePentraxin 3Tissue factorPAI-1Birc2 Naip1RhobCasp3

FunctionalAnalysis

RemodelingAnalysis

MolecularAnalysis

Vasoactive

Pro-Inflammatory

Oxidant/anti-oxidant

Prothrombotic

Flow Cytometry

Hand vein LVDTconduit veins

Apoptosis

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.

Linear RNA Amplification

Nuclear staining Von Willebrand

10 μm 10 μm

COX-2 COX-2 (digitized and processed)

10 μm

Quantitative Immunofluorescence Microscopy

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

Healthy subjectsCompensated CHFDecompensated CHF

B) COX-2

2

4

6

8

0

*

‡

31

0

1

3

iNOSD)

2 †§

C) eNOS

0.5

1

1.5

2

0

1 2

1

3

A) Nitrotyrosine

0

1

2

3

4

5

31 2

*

‡

Colombo PC. Circulation. In press.Colombo PC. Circulation. 111:58, 2005

Endothelial Activation Quiescence