Download Endothelial Cells - Gastaldi Congressi

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 20gauge 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 CD146
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
CD146
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-kB-IkBα
NF-kB
IkBα
endothelial cell
Endothelial activation
In Inflammatory disease
pro-inflammatory genes
COX-2
iNOS
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
OXIDATIVE STRESS !
•Hypertension
•Hypercholesterolaemia
•Diabetes
NO balance
•Smoking
•CAD
•CHF
Endothelial Dysfunction!
Linke A, e al. Heart Failure Reviews, 2003;8:87
Nitrotyrosine
COX-2
7000
6000
5000
4000
3000
2000
1000
0
3000
2500
2000
1500
1000
500
0
1
2
3
1
Artery decompensated CHF
2
3
Vein controls
Vein decompensated CHF
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 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 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
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
Compensated 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
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
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
ProInflammatory
Prothrombotic
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
FMD
conduit arteries
Endo-PAT2000
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 (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
Quantitative Immunofluorescence Microscopy
10 μm
Nuclear staining
10 μm
Von Willebrand
10 μm
COX-2
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
2
A)
Nitrotyrosine
2
‡
5
1.5
4
pixel ratio
pixel ratio
1.5
*
3
1
2
1
0.5
1
2
4
1
*
2
3
0
2
C) eNOS
1
2
D) iNOS
1
0
2
3
1
3
2
3
3
2
1.5
§
1.5
1.5
pixel ratio
pixel ratio
6
0.5
0
0
1
‡
8
1
1
0.5
0.5
2
†
1
1
0.5
0
0
1
2
3
0
1
Decompensated
CHF
0
2
3
Compensated
CHF
Endothelial Activation
1
Healthy subjects
2
Quiescence
Colombo
PC.
Circulation.
In 111:58,
press. 2005
Colombo
PC.
Circulation.
3