Endothelial function.aha.
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This document discusses markers of endothelial function and their relationship to cardiovascular risk and disease. It describes how endothelial dysfunction, measured by reduced flow-mediated dilation or impaired acetylcholine-induced dilation, predicts future cardiovascular events independent of traditional risk factors. Oxidative stress and reduced endothelial progenitor cell counts are also associated with endothelial dysfunction and increased risk. Improvement in endothelial dysfunction through treatment is correlated with reduced cardiovascular risk, showing it is an important therapeutic and prognostic target.
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Endothelial function.aha.
- 1. Systemic markers of endothelial function Arshed A. Quyyumi M.D. Professor of Medicine Emory Univesrity School of Medicine
- 2. ATHEROSCLEROSIS Cardiovascular events RISK FACTORS OXIDATIVE STRESS ENDOTHELIAL DYSFUNCTION INFLAMMATION
- 3. ENDOTHELIUM SMOOTH MUSCLE PLATELETS - - Shear Stress 2 EDHF PGI L-NMMA - Relaxation GTP cGMP Guanylate cyclase (inactive) Nitric Oxide Guanylate cyclase (active) L-ARGININE Nitric Oxide NOS L-citrulline R BradykininAcetylcholine
- 4. Endothelium-dependent probes: •Acetylcholine, •Bradykinin, •Substance P, •L-NMMA, •Shear Stress Endothelium- independent probes: •Sodium nitroprusside, •Adenosine Measurements: •Coronary blood flow and resistance •Epicardial Diameter Assessment of Coronary Vascular Function
- 9. Brachial artery reactivity • Flow-mediated vasodilation (FMD) – Endothelium-dependent • Baseline measurements • Reactive Hyperemia • Sublingual Nitroglycerin response (NTG) – Endothelium-independent RestRest ReactiveReactive HyperemiaHyperemia
- 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
- 13. Endothelial dysfunction No atherosclerosis • Sedentary lifestyle •Hypertension • Hypercholesterolemia • Diabetes • Heart failure • Estrogen withdrawal • Age • Smoking • Homocystinuria • Prinzmetal's angina Atherosclerosis Platelet activation tPA ↓ , PAI-1 ↑ •thrombosis •Adhesion molecule expression •Cytokine release •Growth factors ↑ •progression of atherosclerosis Abnormal vasomotion •myocardial ischemia •coronary spasm •hypertension
- 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
- 16. Event-Free Survival According to Coronary Vascular Function (Death, myocardial infarction, unstable angina, stroke) Acetylcholine Highest tertile Mid tertile Lower tertile Months 0 12 24 36 48 60 72 84 96 EventFreeSurvival(%) 0 50 60 70 80 90 100 Microvascular vasodilation Greatest Intermediate Least Vasodilation p=0.047 Months 0 12 24 36 48 60 72 84 96 EventFreeSurvival(%) 0 50 60 70 80 90 100 Dilation Constriction Epicardial changes p=0.003 Halcox J, Quyyumi A Circulation 2002 308 patients
- 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
- 22. ATHEROSCLEROSIS Cardiovascular events RISK FACTORS OXIDATIVE STRESS ENDOTHELIAL DYSFUNCTION INFLAMMATION
- 23. ROS O2 ·- H2O2 HOCl OONO- NO LO· LOO· HO· Glutathione GSH GSSG GPX H2O2 NO2- Ox-Hb met-HbNO-Hb NO3- LDL Apo B E E E E E Protein oxidation Lipid oxidation LOH LOOH LONO LOONO Isoprostanes TBARS HNE MDA oxLDL oxLDL Aby’s Cysteine Cystine
- 24. Endothelial function and markers ofEndothelial function and markers of oxidative stressoxidative stress 126 healthy non-smokers EhCys/CySS -73 -72.5 -72 -71.5 -71 -70.5 -70 -69.5 -69 -68.5 -68 FMD <7% FMD >7% Endothelial Function P=0.03 -72.7±8 -69.7±8 Cysteine redox 290 300 310 320 330 340 350 360 370 380 FMD <7% FMD >7%FORT(Carrunits) Endothelial Function P=0.02 377±20 319±17 FORT
- 25. EPCs and vascular diseases Risk Factors Endothelial injury Endothelial dysfunction, Inflammation AtherogenesisAtherogenesis Vascular RepairVascular Repair Endothelial Progenitor CellsEndothelial Progenitor Cells Oxidative stress
- 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
Editor's Notes
- 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 .