10. CP914474- 11
10 min 5 min 10 min 10 min
Cuff inflation
60 mm Hg > systole
pressure
Cuff
deflation
NTG
SL
Pulsatile arterial tonometry (PAT)Pulsatile arterial tonometry (PAT)
CP989904-9
11. CP914474- 12
PAT Hyperemic Response – Mayo StudyPAT Hyperemic Response – Mayo Study
Compared to Intra Coronary Blood Flow Response to AcetylcholineCompared to Intra Coronary Blood Flow Response to Acetylcholine
N=94N=94
1
1.2
1.4
1.6
1.8
2
Abnornmal coron resp Normal coron resp
PATIndex
p<.00001n=55
n=39
Mayo Clinic (Abnormal if CBF<50% or CAD<-20%) Lerman et al
12. CP914474- 13
The Effect of Nitric
PWAFollowing Re
0
20
40
60
80
100
120
140
160
180
1 2 3 4 5
Control
L-NAME
14. Endothelial function is an independentEndothelial function is an independent
determinant of long term prognosisdeterminant of long term prognosis
15. CP914474- 17CP1137788-2
-8.0 -6.0 -4.0 -2.0 0.0 2.0 4.0 6.0 8.0
Suwaidi et al, 2000
Schanginger et al, 2000
Neunteufl et al, 2000
Perticone et al, 2001
Heitzer et al, 2001
Halcox et al, 2002
Modena et al, 2002
Schindler et al, 2003
Gokce et al, 2003
Targonski et al, 2003
Relative risk: Dysfunction
vs. normal function
Prediction of future cardiovascular eventsPrediction of future cardiovascular events
by measurement of endothelial functionby measurement of endothelial function
17. CAD
Event-Free Survival According to
Coronary Vascular Function
308 patients undergoing catheterization with and without CAD/follow-up 44±2 months
Events: Death, CVA, Myocardial infarction, Unstable angina
Normal Coronaries
Months
0 12 24 36 48 60 72 84 96
Event
Free
Survival
(%)
0
20
40
60
80
100
Highest
Mid + Lowest
Tertiles of Acetylcholine Response
p=0.035
Halcox, Quyyumi, Circulation 2002
18. Endothelial function and risk of developing
hypertension
952 healthy post menopausal
women
Age 44 to 60 years
Baseline normal BP
Follow-up for mean 3.6 years
Results:Results:
112 developed hypertension
Relative risk of developing HTN
during follow-up was 5.8 fold in
those in the lowest FMD tertile
compared to the highest tertile.
Rossi R J Am Coll Cardiol 2004;44:1636
19. Endothelial function and risk of developing
diabetes
FMD tertile Highest Mid Lowest
FMD % >5.6 4.4-5.5 <4.3
Incident
diabetes
9 35 58
Incident
rate (per 1000
person years)
2.0 7.9 14.4
Multiple
adjusted
RR
1 2.85 5.4
840 healthy non-obese post-
menopausal women
Mean age 53 years
Baseline normal glucose and
OGTT
Follow-up for mean 3.9 years
Results:Results:
102 developed Type II diabetes
Relative risk of developing
diabetes during follow-up was
5.9 fold in those in the lowest
FMD tertile compared to the
highest tertile.
Rossi R J Diabetes Care 2005;2: 702
20. Endothelial Function Predicts Future Development
of Coronary Artery Disease
• >10 year FU of 42 women with chest pain, normal
CAs, positive SPECT thallium scans, who also
underwent endothelial function testing
• 22 patients had vasoconstriction and 20 dilation with
acetylcholine
• In ACH + group; 1 death, 13 continued pain
•13 patients in the ACH + group developed CAD by
angiography
• In ACH – group; resolution of CP after 6 to 36 months,
SPECT negative, no CAD by angiography after 10 years
Bugiardini Circulation 2004;109;2018
21. Is improvement in endothelial dysfunction an
indicator of improved prognosis?
0
2
4
6
8
10
12
14
16
18
20
CVE Hosp/CHF TIA's
Δ FMD < 10%*
Δ FMD >10%*
* change in FMD from baseline
** no difference in type of Rx or end Rx BP Modena et al, JACC, 2002
400 hypertensive post-menopausal women
Responders: FMD improved >10% (4 to 7.1%) in 250
Non-responders: FMD change <10% (4 to 4.1%) in 150
26. Endothelial Progenitor Cell
Colony Forming Assay
• Measure of progenitor cell
capacity
• Density gradient
separation of PBMCs
• Fibronectin plate
– 1million cells per well/24
well
• Colony counts after 7days
• Simple and reproducible Hill, Quyyumi, Finkel N Engl J Med 2003;13:593
27. Endothelial Progenitor Cell Colony Formation
and Cardiovascular Risk Profile
EPC-CFUEPC-CFU
Framingham Risk ScoreFramingham Risk Score
Hill, Quyyumi, Finkel N Engl J Med 2003;13:593
45 Healthy Males, >21 years (mean age 50.3±1.7) without cardiovascular diseases
28. Relation between endothelium-
dependent function and EPCs
EPC-CFUEPC-CFU
Flow mediated dilation –FMDFlow mediated dilation –FMD
Hill, Quyyumi, Finkel N Engl J Med 2003;13:593
45 Healthy Males, >21 years (mean age 50.3±1.7) without cardiovascular diseases
RestRest
ReactiveReactive
HyperemiaHyperemia
•Flow-mediated vasodilation (FMD)
EPC-CFUEPC-CFU
29. Systemic markers of endothelial
function
Arshed A. Quyyumi M.D.
Professor of Medicine
Emory Univesrity School of Medicine
Notas del editor
C:\My Documents\RC graphics\1997\11 Nov Talk\Talk.pr4
Flow-mediated vasodilation(FMD) or the endothelium dependent response The response to sublingual nitroglycerin spray to asses the endothelium-independent vasodilation was then measured.
Studies were performed by a single experienced operator.
was assessed by measuring the maximum increase in diameter of the brachial artery during reactive hyperemia created by a cuff inflated at suprasystolic pressure for 5 mins. After rapid cuff deflation, flow velocity is measured and the artery lumen was imaged continually for the next 120 seconds of hyperemia.
Here you can appreciate the difference in diameter between rest and during reactive hyperemia.
Figure 1. Relative risk of developing hypertension in relation to the percent of flow-mediated dilation (FMD) quartiles. The fourth quartile must be considered as the referent one. p &lt; 0.0001 for trend. Numbers in the bars represent the incidence rate (new cases of hypertension per 1,000 person-years). Q = quartile.
Russell Ross’ classic paradigm argues that seemingly disparate risk factors such as hypertension, increased serum cholesterol, or diabetes function through a final common pathway that culminates in endothelial cell injury. This concept has now been expanded to include both endothelial dysfunction as well as endothelial damage. Consistent with this interpretation are a number of recent studies demonstrating that endothelial function is a predictor of future cardiovascular events. Cardiovascular risk factors thus contribute to atherogenesis by inducing endothelial cell injury and dysfunction.
Cardiologists are now familiarizing themselves with the new biology of bone marrow stem cells. Endothelial progenitor cells or EPCS are one such group of cells which many groups have been investigating for their potential role in angiogenesis. However given their relatively high frequency in the circulation and the limited degree of neovascularization occurring in healthy adults, EPCs may well have an important role beyond angiogenesis. We have become interested in EPCs and the concept that endothelial injury may be opposed by a limited ability of the vessel wall to undergo repair.
Endothelial progenitor cell isolation and colony forming assay
There a number of ways to measure EPC numbers and function. We have previously used immunhistochemical identification with endothelial specific markers such as VEGF Receptor 2/KDR, CD31 or TIE2. More recently there have been flow cytometric methods for identifying cells which express a stem cell marker such as CD34 or AC133 as well as an endothelial specific marker.Funtional assessments of migratory capacity can be done using cytokines such as vascular endothelial growth factor. Staining using fluorescent acetytlated low density lipoprotein as shown here can also be used.
In this study we have measured a colony forming assay as a marker of EPC activity. We believe that colony formation is an indicator of the stem or progenitor cell capacity of these circulating cells.
Peripheral blood mononuclear cells are isolated uisng density gradient centrifugation. The cells are washed and preplated on fibronectin for 48 hours to remove mature circulating endothelial cells and the non adherent fraction plated at relatively high density on 24 well fibronectin coated plates. After 7 days the colonies which form can then be counted in a simple and reproducible manner.. For the purposes of this study An endothelial progenitor cell colony consisted of multiple thin, flat cells emanating from a central cluster of rounded cells. A central cluster alone without associated emerging cells was not counted as positive. Colonies were counted manually in a minimum of four separate wells by observers unaware of the clinical profile. Confirmation of endothelial lineage was performed in selected subjects.
.
For measurement of cellular senescence, we recruited from the original 45 subjects a subset of 16 age-matched patients who were divided into high and low Framingham risk groups (mean 7.3 +/- 2.3 versus 1.5+/- 2.1; p&lt;0.01). Endothelial progenitor cell cultures derived from these individuals were maintained for 7 days with fresh media supplied every 3 days. Senescence associated ß-galactosidase (SA- ß-Gal) activity was measured as previously described in Dimri et al. 25. Briefly, cells were fixed with 4% formaldehyde and incubated overnight in X-Gal solution (1 mg/ml) at 37 oC without CO2. Only isolated cells away from central colonies were analyzed and only those cells with distinct blue cytoplasmic color were counted as positive. The percentage of positive cells was determined by counting four random fields containing approximately 100-200 cells.
We next assessed whether the level of circulating endothelial progenitor cells correlated with the presence or absence of known conventional cardiovascular risk factors. As noted here, endothelial progenitor cell colony forming units were significantly reduced in patients with elevated serum cholesterol level. We also observed a negative correlation between the patient’s age and circulating endothelial progenitor cells, however this relationship was not statistically significant.
To determine whether a cumulative risk factor profile influenced EPC counts, we calculated the Framingham risk factor score for each patient and found a significant inverse correlation between the calculated risk score and EPCs, with higher scores associated with diminished endothelial progenitor cell counts
In addition to quantitative differences in EPCS in patients with high and low cardioavascular risk scores we hypothesized that their may also be qualitative differences. We examined 16 age matched subjects with high or low risk scores and measured Endogenous beta galactosidase activity which is widely used marker of cellular senescence. Even in this small number of patients there was a significant difference in cellular senescence thus patients at high risk have both fewer EPCS and EPCs which appear biologically older.
Because the vascular endothelium integrates the injury from established and as yet unknown risk factors, we next assessed the relationship between endothelial progenitor cell colony counts and flow mediated brachial reactivity, a composite measure of endothelial integrity. As noted here, there was a strong correlation between endothelial progenitor cell colony count and flow mediated brachial reactivity. When the measured FMD was divided into tertiles (Click) subjects with the highest flow mediated brachial reactivity had endothelial progenitor cell colony counts approximately 3-fold higher than those in the lowest tertile .