WO2009152619A1

WO2009152619A1 - A method of detecting a subject at risk of, or having, an indication associated with coronary artery disease

Info

Publication number: WO2009152619A1
Application number: PCT/CA2009/000854
Authority: WO
Inventors: Jean-Claude Tardif; Berry Colin; Eric Rheaume
Original assignee: Institut De Cardiologie De Montréal; University Court Of The University Of Glasgow
Priority date: 2008-06-17
Filing date: 2009-06-17
Publication date: 2009-12-23

Abstract

There are disclosed methods and biomarkers for detecting an indication associated with, or risk of, coronary artery disease In an embodiment, a method includes obtaining a sample including bone marrow-derived cells and a growth factor; measuring a ratio of cell count of the cells and concentration of growth factor, and determining, based on the ratio of the cells and the concentration of the growth factor, risk of coronary artery disease In another embodiment, a biomarker includes a ratio of a cell count of bone marrow-deπved cells and a concentration of a growth factor, measured in a biological sample including the bone marrow-derived cells and the growth factor, the ratio of the cell count of the bone marrow-derived cells and the concentration of the growth factor measured in the sample, determining whether the subject has the indication associated with coronary artery disease Other embodiments are also disclosed.

Description

A METHOD OF DETECTING A SUBJECT AT RISK OF, OR HAVING, AN INDICATION ASSOCIATED WITH CORONARY ARTERY DISEASE

Reference To Pending Prior Patent Application

This application claims the benefit under 35 U. S. C. 119 (e) of U.S. Provisional Patent Application Number 61/129,305, filed June 17, 2008 by the inventors of the present Patent Application for "Human Coronary Atherosclerosis, Vascular Progenitor Cell Death Susceptibility, Circulating Number and Ratio to Serum Vascular Endothelial Growth Factor," which patent application is hereby incorporated herein by reference.

Background

[001] Endothelial progenitor cells (EPCs) contribute to vascular regeneration and repair, and may have anti-atherosclerotic actions. There is conflicting information in the prior art whether reduced circulating EPC numbers are associated with severity of coronary artery disease (CAD). Furthermore, the prior art does not indicate whether deficiencies in EPC number and/or functionality should be implicated in CAD, or whether these abnormalities might be a cause or a consequence of an enhanced CAD burden.

[002] One important limiting factor in studies of progenitor cell function in CAD relates to imaging. Previous investigations have related visual angiographic estimates of CAD severity to measures of EPC number and function. However, this approach has several limitations. While coronary angiography provides insights into lumen loss, it fails to inform on the coronary artery wall and atheroma burden. By contrast, intravascular ultrasound (IVUS) provides a volumetric quantitative measurement of coronary atherosclerosis which in turn correlates with the global burden of CAD severity. IVUS is the current gold standard for quantification of coronary atheroma, yet to date, studies of progenitor cell biology have failed to quantify atherosclerosis with this stringent technique.

[003] A definitive consensus on the definition of EPCs is still elusive, probably due to the diverse origins of cells with capacities to be involved in endothelium maintenance and initiation of neovascularization or capillogenesis. Circulating EPCs are enriched in a mononuclear cell population that expresses the cell surface antigens CD34 and KDR. Although the mononuclear cell population obtained after selection for fibronectin adhesion and cultured under endothelial cell promoting conditions does not usually predominantly express CD34 and KDR, these cells have an important role in neovascularization through mechanisms which include vascular endothelial growth factor (VEGF) secretion. Such cultured cells may be referred to as early outgrowth culture-expanded EPCs (CE-EPCs). These cells are bi-phenotypic in that they co-express both myeloid and endothelial markers.

[004] Summary of the Invention

[005] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key aspects or essential aspects of the claimed subject matter. Moreover, this Summary is not intended for use as an aid in determining the scope of the claimed subject matter.

[006] In an embodiment, there is provided a method of detecting in a subject an indication associated with coronary artery disease, the method including obtaining a biological sample including circulating endothelial progenitor cells (EPCs) and vascular endothelial growth factor (VEGF); measuring a ratio of a cell count of the EPCs and a concentration of the VEGF in the sample; and determining, based on the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample, whether the subject has the indication associated with coronary artery disease.

[007]For example, the biological sample is a blood sample, and, in some embodiments, the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

[008] In another embodiment, there is provided a method of detecting a subject at risk of coronary artery disease, the method including obtaining a biological sample including circulating endothelial progenitor cells (EPCs) and vascular endothelial growth factor (VEGF); measuring a ratio of a cell count of the EPCs and a concentration of the VEGF in the sample; and determining, based on the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample, the risk of coronary artery disease.

[009] In yet another embodiment, there is provided a method of detecting in a subject an indication associated with coronary artery disease, the method including obtaining a biological sample from the subject including bone marrow- derived cells and a growth factor; measuring a ratio of a cell count of the bone marrow-derived cells and a concentration of the growth factor in the sample; and determining, based on the ratio of the bone marrow-derived cells and the concentration of the growth factor measured in the sample, whether the subject has the indication associated with coronary artery disease.

[0010] For example, the bone marrow-derived cells are circulating endothelial progenitor cells (EPCs), leucocytes, platelets, or any other suitable bone marrow- derived cells. In some embodiments, the growth factor is vascular endothelial growth factor (VEGF). In some embodiments, the step of measuring the cell count of the bone marrow-derived cells is determined with flow cytometry or an automated blood cell counter.

[0011] In still another embodiment, there is provided a method of detecting a subject at risk of coronary artery disease, the method including obtaining a biological sample from the subject including bone marrow-derived cells and a growth factor; measuring a ratio of a cell count of the bone marrow-derived cells and a concentration of the growth factor in the sample; and determining, based on the ratio of the bone marrow-derived cells and the concentration of the growth factor measured in the sample, the risk of coronary artery disease. [0012] In another embodiment, there is provided a biomarker for detecting in a subject an indication associated with coronary artery disease, the biomarker including a ratio of a cell count of circulating endothelial progenitor cells (EPCs) and a concentration of vascular endothelial growth factor (VEGF), measured in a biological sample including the EPCs and the VEGF, the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample, determining whether the subject has the indication associated with coronary artery disease.

[0013] In another embodiment, there is provided a biomarker for detecting a subject at risk of coronary artery disease, the biomarker including a ratio of a cell count of circulating endothelial progenitor cells (EPCs) and a concentration of vascular endothelial growth factor (VEGF), measured in a biological sample including the EPCs and the VEGF, the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample determining the risk of coronary artery disease.

[0014] In yet another embodiment, there is provided a biomarker for detecting in a subject an indication associated with coronary artery disease, the biomarker including a ratio of a cell count of bone marrow-derived cells and a concentration of a growth factor, measured in a biological sample including the bone marrow- derived cells and the growth factor, the ratio of the cell count of the bone marrow- derived cells and the concentration of the growth factor measured in the sample, determining whether the subject has the indication associated with coronary artery disease.

[0015] In still another embodiment, there is provided a biomarker for detecting a subject at risk of coronary artery disease, the biomarker including a ratio of a cell count of bone marrow-derived cells and a concentration of a growth factor, measured in a biological sample including the bone marrow-derived cells and the growth factor, the ratio of the cell count of the bone marrow-derived cells and the concentration of the growth factor measured in the sample determining the risk of coronary artery disease. [0016] The present Application cites many documents, the contents of which is hereby incorporated by reference in its entirety.

[0017] Other embodiments are also disclosed. Additional objects, advantages and novel features of the technology will be set forth in part in the description which follows, and in part will become more apparent to those skilled in the art upon examination of the following, or may be learned from practice of the technology.

[0018] Brief Description of the Drawings

[0019] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0020] Figure 1 illustrates relationships between percent atheroma volume measured by IVUS and the change of total necrosis rate of CE-EPCs induced by CRP exposure (A, n=41 ) and between coronary artery score measured by QCA and the fold change of CE-EPC total cell death (sum of apoptosis and total necrosis rates) following CRP exposure (B, n=41 ).

[0021] Figure 2 illustrates relationships between percent atheroma volume measured by IVUS in stable patients and circulating absolute blood count of EPCs (A, n=20) and the ratio of serum VEGF concentration (pg/mL) to the absolute number of EPCs (CD34/KDR double-positive cells) per μL of blood (B, n=19).

[0022] Figure 3 illustrates relationships between serum HDL-cholesterol concentration in stable CAD patients and circulating CD34/KDR double-positive (A, n=19) or CD34-positive (B, n=26) cell numbers.

[0023] Detailed Description

[0024] Embodiments are described more fully below in sufficient detail to enable those skilled in the art to practice the system and method. However, embodiments may be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. The following detailed description is, therefore, not to be taken in a limiting sense.

[0025] As EPCs appear to have pathophysiologic importance in CAD. First, the relationship between circulating EPC numbers and quantitative measurements of human coronary atherosclerosis is considered. Second, the relationships between one of the main stimuli for EPC release from the bone marrow, VEGF, circulating EPCs, and human coronary atherosclerosis is considered. Third, the consequences of reduced EPC function, such as impaired EPC survival, in the pathogenesis of CAD is considered. Fourth, since an acute exacerbation of systemic inflammation is a distinguishing characteristic of acute coronary syndromes (ACS) compared to stable CAD, the influence of inflammation on CE- EPC functionality is considered.

[0026] To explore the above-identified considerations, circulating EPC numbers were determined and we studied the in vitro functions of CE-EPCs in patients with stable or unstable CAD, and quantified coronary atherosclerosis using IVUS. This assessment of global CAD severity was complemented with computer-assisted quantitative coronary angiography (QCA). Observations confirm a link between CE-EPC susceptibility to cell death and plaque burden by IVUS and QCA, and these relationships were particularly pronounced in ACS patients. In stable patients, the ratio of VEGF concentration to circulating EPC number correlated strongly with atheroma volume, and this ratio may be indicative of the effectiveness of VEGF-induced EPC mobilization from the bone marrow to the circulation in stable CAD. The ratio of VEGF to circulating EPC count is a sensitive biomarker for CAD severity in stable patients.

r0027T METHODS

[0028] Patients with a contraindication to contrast injection (known allergy to iodinated contrast agents, renal insufficiency), heart failure, prior coronary revascularization, severe concomitant illness or severe coronary artery calcification, were excluded. A total of 66 patients gave informed consent for this investigation. Of these, eight patients were subsequently found to have coronary anatomy unfavorable for IVUS examination and four other patients provided blood samples which were inadequate for analyses (e.g. insufficient sample volume). Fifty four patients had coronary imaging and basic laboratory data. Of these, 27 consecutive stable patients aged 53 to 69 years, with a history of suspected ischemic chest pain, and scheduled for clinically indicated non-urgent coronary angiography were included. Twenty seven patients aged 39 to 77 years with an indication for coronary angiography within one week of an ACS event defined as unstable angina, non-ST-segment or ST-segment elevation myocardial infarction (Ml) were also enrolled.

[0029] The methods of the IVUS procedure have been detailed previously (see for example "Berry C, L'Allier PL, Gregoire J, et al. Comparison of intravascular ultrasound and quantitative coronary angiography for the assessment of coronary artery disease progression. Circulation 2007; 1 15:1851 -7" and "Tardif JC, Gregoire J, L'Allier PL, et al. Effects of the acyl coenzyme A:cholesterol acyltransferase inhibitor avasimibe on human coronary atherosclerotic lesions. Circulation 2004; 1 10:3372-7"). In brief, single-vessel IVUS studies were performed using 40 MHz IVUS catheters (Boston Scientific). Intra-coronary nitroglycerin (150 μg) was administered before the IVUS examination. The IVUS catheter was advanced distally, at least 40 mm beyond the coronary artery ostium, to a recognizable landmark (arterial branch). The transducer was then pulled back automatically at a speed of 0.5 mm/s up to the guiding catheter with the use of a validated motorized device. A detailed running audio commentary was recorded during the pullback. A second pullback was then performed in the same coronary artery using the same guidelines to ensure high-quality imaging. The proximal 4 cm of the target coronary artery in which IVUS was performed needed to have a reference diameter of 2.5 mm or more, be free of filling defects suggestive of thrombus, not have more than a 50% reduction in lumen diameter by visual angiographic estimation, and not have undergone previous percutaneous coronary intervention nor have been a candidate for intervention at the time of the catheterization. [0030] IVUS image analyses were performed according to standardized methods that have previously described (for example, see Berry et al., cited previously). All IVUS examinations were analyzed at the Montreal Heart Institute IVUS Core Laboratory by experienced technicians supervised by a cardiologist blinded to EPC results, according to published standards (Mintz GS, Nissen SE, Anderson WD, et al. American College of Cardiology Clinical Expert Consensus Document on Standards for Acquisition, Measurement and Reporting of Intravascular Ultrasound Studies (IVUS). A report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents. J Am Coll Cardiol 2001 ; 37:1478-92.16). Quantitative IVUS analyses were performed using a custom- developed system and software for geometric computations (Echo plaque, INDEC Systems, California). Coronary artery lumen and external elastic membrane (EEM) borders were traced manually on digitized cross-sections at every 1 mm in the 30-mm segment of interest. Measurements of lumen, plaque and EEM areas were available for every traced cross-section of the analyzed segment. Percent atheroma volume was calculated by dividing plaque volume by total vessel volume and then multiplying by 100. Percent atheroma volume determined the proportion of external elastic membrane (EEM) volume occupied by atheroma. This measure correlates with the global severity of CAD in all coronary arteries when measured by QCA analyses. The intra-class correlation coefficients for inter-observer variability of measurement of plaque and vessel volumes in our laboratory are 0.98 and 0.99.

[0031] Angiographic procedure and analyses were used as follows, lntracoronary nitroglycerin (150 μg) was administered into each coronary artery before angiographic examination. The segments of interest were visualized in multiple transverse and sagittal views to clearly separate stenoses from branches, minimize foreshortening, and obtain views as perpendicular as possible to the long-axis of the segments to be analyzed. All coronary angiograms were analyzed at the Montreal Heart Institute QCA Core Laboratory by means of the Clinical Measurements Solutions system (QCA-CMS, Version 5.1 ; MEDIS Imaging Systems, Leiden, The Netherlands). QCA was performed by experienced technicians supervised by an expert physician blinded to EPC data. For each lesion, an end-diastolic frame was selected that best showed the stenosis at its most severe degree with minimal foreshortening and branch overlap. The coronary artery segments analyzed included all those with a reference diameter 1.5 mm and a stenosis at about 20%. The quantification system has been shown to measure coronary dimensions from different end- diastolic frames with an average standard deviation (SD) of the measurement differences of 0.13 mm (see "Waters D, Lesperance J, Craven TE, Hudon G, Gillam LD. Advantages and limitations of serial coronary arteriography for the assessment of progression and regression of coronary atherosclerosis - implications for clinical-trials. Circulation 1993; 87:1138-47."). Computer software automatically calculates the minimum lumen diameter (MLD), reference diameter, percentage diameter stenosis and stenosis length.

[0032] QCA measures of CAD severity were made as follows. The QCA variables included the following: 1) the cumulative coronary stenosis score, calculated by adding all percent diameter stenoses in SI units (50%=0.50). The cumulative coronary stenosis score is an index of the anatomic extension and severity of CAD, and a higher value reflects more severe disease ("Solymoss BC, Bourassa MG, Campeau L, et al. Effect of increasing metabolic syndrome score on atherosclerotic risk profile and coronary artery disease angiographic severity. Am J Cardiol 2004; 93:159-64."); 2) the global plaque area score was calculated by averaging all of the plaque area values in the segments studied. The plaque area by QCA is represented by the area between the estimated interpolated reference and luminal contours within the obstruction boundaries; 3) the coronary artery score represents the per-patient average of the MLD of all measured segments; a higher score reflects less obstructed coronary artery lumen ("Waters D, Higginson L, Gladstone P, et al. Effects of monotherapy with an HMG-CoA reductase inhibitor on the progression of coronary atherosclerosis as assessed by serial quantitative arteriography - the Canadian coronary atherosclerosis intervention trial. Circulation 1994; 89:959-68.19").

[0033] Analysis of endothelial progenitor cells

[0034] A 40 mL venous blood sample was obtained (i.e., the blood sample collection) from recumbent patients prior to angiography. Five mL were used to obtain serum and the remainder of the blood sample was placed in potassium EDTA anti-coagulant and transferred for immediate laboratory analyses.

[0035] EPC quantification (in whole blood) was performed according to a modification of the ISHAGE guidelines for determination of blood CD34+ cell count by flow cytometry ("Sutherland DR, Anderson L, Keeney M, Nayar R, Chin- Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother 1996; 5:213-26."). Briefly, 100 μl_ of blood was immunostained with the Stem-Kit (TM) (Beckman Coulter) monoclonal antibodies: a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to the human CD45 antigen and a phycoerythrin (PE) conjugated CD34 antibody were used, to which was added a monoclonal antibody to human KDR (Abeam) conjugated with Zenon Alexa Fluor 647-RPE labeling kit (Invitrogen). Isotype-identical antibodies served as controls. After incubation, erythrocytes were lysed in NH₄CI solution. Stem-Count fluorospheres from the Stem-Kit (TM) were added to each sample for the direct determination of absolute EPC count per μl_ of blood. Flow cytometry was performed in a Beckman Coulter EPICS XL flow cytometer. The gating strategy for CD34 and for EPCs selected for CD45dim and excluded CD45 bright cells. EPC quantification was performed in triplicate for each patient. Results are expressed as mean absolute numbers of CD34 (n=27 stable and 25 unstable patients) and CD34/KDR (n=20 stable and 22 unstable patients) double-positive cells per μl_ of whole blood.

[0036] Early outgrowth culture-expanded EPCs (CE-EPCs) is one method used for culturing cells. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation. PBMCs were plated on fibronectin-coated plates (BD Biosciences) at a density of 1.5 x 10⁶/cm² and cultured at 37⁰C under 5% CO₂ in EGM-2 (Lonza) supplemented with 20% ES cell-qualified fetal-bovine serum (Mexico) (Invitrogen). After 4 days in culture, non-adherent cells were removed by washing and the remaining cells were kept in culture for 3 additional days.

[0037] Quantification of basal and C-reactive protein (CRP)-stimulated CE-EPC cell death (apoptosis or necrosis) was determined by cytometry analysis of Annexin V- and propidium iodide (Pl) stained cells. After 7 days in culture, CE- EPCs were washed and incubated at 37⁰C for 3h with EGM-2 (0.5% serum) with or without 225 mg/L of human CRP purified from plasma (Trichem Resources Inc) to measure susceptibility to cell death induced by CRP at concentrations in the upper range of those that can be observed in high-risk patients following acute Ml. After incubation, detached cells in the media were pooled with cells that were harvested by mild treatment with dispase 0.5 mg/mL and gentle pipetting, stained with Annexin V and Pl (BD Biosciences) and analyzed by flow cytometry. Apoptotic, late apoptotic, necrotic and total necrotic cells are defined here as cells positive for Annexin V only, cells positive for both Annexin V and Pl, cells positive for Pl only and all cells positive for Pl (late apoptosis and necrosis), respectively. Apoptosis and/or necrosis counts (percentage of Annexin V and/or Pl positive cells on total CE-EPCs) with and without CRP incubation were obtained in 19 stable patients and 22 unstable patients.

[0038] Basal CE-EPC nitric oxide (NO) secretion was evaluated by the non cell- permeant 4-amino-5-methylamino-2',7' difluorofluorescein (DAF-FM) fluorescent NO indicator (Invitrogen) according to slight modifications of the method described by Rathel et al in "Rathel TR, Leikert JJ, Vollmar AM, Dirsch VM. Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cells in vitro. Biol Proced Online 2003; 5:136-42". On the 6th day of culture, CE-EPCs from 3 different wells were washed and incubated in dark- adapted conditions for 24 h in DAF-FM solution (0.1 μM) in EBM-2 (0.5% serum) at 37¹C under 5% CO₂. Non-adherent cells were then pelleted by centrifugation. Measurement of fluorescence in supernatant was performed at 37³C using a QPCR system as a 96-well plate reader (Mx3005P, Stratagene) with an excitation wavelength of 492 nm and an emission wavelength of 516 nm (FAM filter set). Secretion of NO in the medium was estimated by comparing fluorescence values to those obtained from a standard curve generated using the NO donor MAHMA NONOate (Alexis Biochemicals). The results were then expressed as equivalent attomoles of NO produced per cell. NO production per CE-EPC was estimated in 22 stable patients and 17 unstable patients. [0039] The number of early outgrowth CE-EPC colonies in culture was counted at day 4 from 10 independent fields at 4X magnification on an Axiovert 200M (Zeiss) microscope for all 27 stable patients and for 24 unstable patients. The counts were presented as the average number of CE-EPC colonies per low power field. A colony was defined as a core of round cells with spindle shaped cells at the periphery. Cellular morphology was confirmed at 10X magnification.

[0040] After 7 days in culture, CE-EPCs were harvested with PBS-EDTA (1 mM) and 2x10⁴ cells were placed on the upper chamber of a 24-well Transwell(TM) migration chamber (Costar, 8 μm pore size) precoated with 50 μg/mL human fibronectin (BD Biosciences). Recombinant human CXCL12 (alias SDF-1 ) (PeproTech, 100 ng/mL in EBM-2/5% FBS) was then added to the lower chamber. All assays were set-up in triplicate. Following a 24-hour migration period, cells were fixed with 10% paraformaldehyde in PBS and stained with 0.1 % crystal violet/20% methanol. Non-migrated cells were carefully removed. Migrated cells, stained blue by crystal violet, were counted at 10X magnification in three random fields per well and the average of each triplicate was calculated. Migration data (control, CXCL12, change in migrated cell number induced by CXCL12 and % induction by CXCL12 versus control) were obtained in 21 stable patients and 15 unstable patients.

[0041] When sufficient CE-EPCs were available after culture and isolation, CE- EPC immunophenotyping characterization was performed. After 7 days of culture, cells were harvested by treatment with PBS-EDTA (1 mM), treated with FcR blocking reagent (Miltenyi) and stained using various antibodies: FITC-conjugated mouse anti-human CD34 (Miltenyi, performed in 10 stable CAD and 4 ACS patients), FITC-conjugated mouse anti-human CD31 (BD Pharmingen, performed in 7 stable CAD and 4 ACS patients), or mouse anti-human KDR (Abeam) followed by addition of PE-conjugated goat anti-mouse IgG (performed in 3 stable CAD and 8 ACS patients). Isotype-matched antibodies were used as negative controls. Labeled cells were analyzed using an EPICS XL flow cytometer (Beckman Coulter).

[0042] Biochemical and hematological profile and serum VEGF measurements. Serum total, HDL and LDL cholesterol, triglycerides and glucose levels were measured with an automated filter photometer system (Dimension RxL Max, Dade Behring, Illinois). Routine hematology was performed by standard methodology using a Coulter Counter STKS (Beckman Coulter) automated cell analyzer. Serum VEGF-A (VEGF165) concentration was determined in 26 stable patients and 26 unstable patients using a human VEGF Quantikine ELISA Kit (R&D systems).

F00431 Statistical analyses

[0044] For continuous variables, depending on the distribution of the data, results are expressed as mean±SD or median (q1 -q3); for categorical variables, results are reported as numbers and percentages. The relationships between EPC number and functions and IVUS or QCA results were analyzed using the Pearson or Spearman correlation test, according to the distribution. CAD characteristics were compared across groups of patients with stable CAD and ACS. For continuous variables, Student's t-test or Wilcoxon test was performed according to the distribution. For categorical variables, the chi-square test was used. P values <0.05 were considered to be statistically significant. All analyses were done with SAS (version 9.1 , SAS Institute, Cary, North Carolina, USA).

r00451 RESULTS

[0046] Clinical, progenitor cell and coronary imaging data were obtained in 54 patients with either a history of stable CAD (n=27) or an ACS (n=27; Table 1 ). None of the patients had a history of arrhythmias, heart failure, or valve disease. Blood samples from unstable patients were obtained 2.9±1.3 days after presentation with ACS. PAV by IVUS was 39.8±9.8% and was similar in stable and unstable patients. PAV correlated with QCA cumulative coronary stenosis score (r=0.55, p<0.0001 ), global plaque area score (r=0.50, p=0.0001 ) and coronary artery score (r= 0.44, p=0.0008).

[0047] Relationships between EPC counts, defined as dual positive CD34/KDR cells in peripheral blood, or CE-EPC colony counts and plaque burden were evaluated. When the groups of stable and unstable patients were combined, neither circulating EPC count nor early CE-EPC colony count correlated with IVUS or QCA CAD measurements (data not shown).

[0048] Flow cytometric analyses demonstrated that CE-EPCs isolated and cultured for 7 days expressed the progenitor cell antigen CD34 (35±11 % positive), the endothelial marker KDR (8±8% positive) and the endothelial/immature myeloid lineage marker CD31 (70±15% positive).

[0049] Correlations were observed between percent atheroma volume on IVUS and CE-EPC total necrosis rate change induced by CRP exposure (n=41 , r=0.40, p=0.01 ; as shown in Fig. 1 , panel A) and the CRP-induced change of late apoptosis rate (n=41 , r=0.35, p=0.025). The QCA cumulative coronary stenosis score correlated with the CRP-induced change in CE-EPC total necrosis rate (n=41 , r=0.33, p=0.035). Coronary artery score on QCA was negatively correlated with the fold change of total cell death (the sum of apoptosis and total necrosis rates) of CE-EPCs induced by CRP exposure (n=41 , r=-0.34, p=0.030; as seen in Fig. 1 , panel B). No significant correlation was detected between basal CE-EPC apoptosis and/or necrosis rates and IVUS or QCA measurement of plaque burden. In the subgroup of patients with CD31 immunophenotyping data, the percentage of CE-EPCs expressing CD31 correlated negatively with the change of CE-EPC total cell death induced by CRP exposure (n=9, r=-0.97, p≤O.0001 ).

[0050] NO production, as estimated by DAF-FM fluorescence per CE-EPC cell, did not correlate with either percent atheroma volume (p=0.72) or with any QCA- derived measure of CAD severity. Induction of CE-EPC migration in response to exposure to CXCL12 (also known as SDF) also did not correlate with percent atheroma volume (p=0.69) or any QCA measurement.

[0051] In stable patients, the absolute number of EPCs per microliter of whole blood correlated negatively with percent atheroma volume on IVUS (n=20, r=-0.50, p=0.023, as sen in Fig. 2, panel A). EPC counts did not correlate with QCA cumulative coronary stenosis score, global plaque area score nor coronary artery score (p=0.8, 0.7 and 0.1 , respectively). VEGF being a critical factor for EPC mobilization, a possible relationship was investigated between the concentration of this protein in serum and the number of circulating progenitor cells. Serum VEGF concentration correlated with the absolute number of CD34+ cells in blood (r=0.39; p=0.049) but not with EPC number (p=0.20).

[0052] As VEGF is a major stimulus for EPC mobilization, it was reasoned that putative deficiencies in vascular repair caused by inappropriate EPC mobilization may be revealed when expressed as the ratio between serum VEGF concentration and circulating EPC number in whole blood. This VEGF/EPC ratio strongly correlated with percent atheroma volume on IVUS of stable patients (r=0.74, p=0.0003, as illustrated in Fig. 2, panel B). The VEGF/EPC ratio also correlated with the global plaque area score on QCA (r=0.52, p=0.024), and tended to correlate with the QCA-derived cumulative coronary stenosis score (r=0.41 , p=0.082). VEGF concentration by itself did not correlate with percent atheroma volume (p=0.20) but did correlate with the global plaque area score (r=0.39, p=0.047) and tended to correlate with the cumulative coronary stenosis score (r=0.35, p=0.079) (Table 2).

[0053] Since VEGF mediates mobilization of many hematopoietic stem cell- derived lineages, it was questioned whether VEGF mobilization of such other lineages could also be altered in relation to coronary plaque burden in stable patients. The relationships of VEGF expressed as a ratio with either leukocytes, monocytes or platelets counts were therefore evaluated. While leucocytes and platelet counts did not correlate with CAD severity, there were significant correlations between the ratio of VEGF/leukocytes and VEGF/platelets for most measurements made from both IVUS and QCA (see Table 2). The coronary artery score on QCA also correlated inversely with monocyte count in stable patients.

[0054] There were differences in CE-EPC findings between patients with stable and unstable CAD. Since the pathogenesis of plaque rupture involves inflammation and vascular cell death, CE-EPC death was next investigated in the groups of patients with stable and unstable CAD. CRP concentrations were 7- fold higher in ACS patients compared to in patients with stable CAD (see Table 1 ). The basal level of CE-EPC necrosis was significantly higher in ACS patients than in stable CAD patients (4.5 (0.41 , 6.23) % vs. 0.49 (0.36, 0.76) %, p=0.004). In ACS patients, percent atheroma volume correlated with the rate change of late apoptosis of CE-EPCs induced by CRP exposure (n=22, r=0.49, p=0.021 ) and the QCA cumulative coronary stenosis score correlated with CRP-induced change of CE-EPC total cell death (n=22, r= 0.57, p=0.006).

[0055] The VEGF/EPC ratio did not correlate with IVUS- or QCA-derived measurements of plaque burden in ACS patients. Serum VEGF concentration was higher in stable patients than in ACS patients (see Table 1 ).

[0056] Relationships between EPCs and risk factors for CAD. When all patients were considered, there was observed an inverse correlation between CE-EPC colony counts and age (n=51 , r=-0.40, p=0.004). In stable CAD patients, serum HDL-cholesterol levels correlated with circulating EPC counts (r=0.67, p=0.002, Fig. 3A), CD34+ cells (r= 0.44, p=0.024, Fig. 3B) and serum VEGF levels (r=0.41 , p=0.037), but not with the VEGF/EPC ratio (p=0.32).

r00571 DISCUSSION

[0058] The observations identified above provide quantitative confirmation of the relationships between vascular progenitor cell number and functions and coronary plaque burden assessed by IVUS and QCA. First, a novel link was demonstrated between CRP-induced augmentation of apoptosis and/or necrosis rate of early outgrowth CE-EPC and human coronary plaque burden. This relationship was strongest in ACS patients. Second, basal level of necrosis of early outgrowth CE- EPC was also significantly higher in unstable compared to stable patients. Third, it was shown that for stable CAD, the ratio of serum VEGF concentration to circulating EPC numbers, defined as the absolute count of dual positive CD34/KDR cells in blood, correlates more strongly with percent atheroma volume as measured by IVUS than the circulating EPC count. The VEGF/EPC ratio also correlates with the global plaque area as measured by QCA. Finally, serum VEGF concentration, circulating CD34-positive cells, and EPC numbers correlated with HDL-cholesterol concentration in stable CAD. [0059] Previous human studies of progenitor cells and coronary atherosclerosis have been limited by the use of visual angiographic estimates of CAD burden. The limitations of this approach, particularly with respect to potential underestimation of disease severity, are well described. In contrast, IVUS enables volumetric measurement of coronary atherosclerosis. For the first time, quantitative information was provided using stringent core laboratory methods definitively linking EPC number and function with the severity of coronary atherosclerosis as determined by both IVUS and QCA.

[0060] It has been shown that greater coronary plaque burden is associated with increased susceptibility of early outgrowth CE-EPCs to CRP-induced apoptosis and/or necrosis. The latter is likely detrimental to the role of EPCs in endothelium repair and vascular maintenance given the pro-inflammatory environment in which EPCs are involved. Importantly, our observations provide mechanistic insights into the causes of EPC deficiency in CAD. Since CAD is characterized by systemic inflammation and increased circulating concentrations of CRP, our observations suggest that the pathogenesis of coronary atherosclerosis and plaque rupture may involve CRP-induced EPC death, leading in turn to reduced vascular repair potential. One possible mechanism for EPC increased susceptibility to cell death could be oxidative stress, since CRP reduces EPC antioxidant defenses. There may be other explanations for the enhanced CE- EPC sensitivity to CRP-mediated apoptosis and/or necrosis. It was observed in the subgroup of patients with immunophenotyping data that CD31 expression correlated negatively with the augmentation of the sum of the apoptosis and necrosis rates of CE-EPCs induced by exposure to CRP. It is known that CD31 can protect cells from apoptosis. Consequently, it is possible that distinct subpopulations of the early outgrowth CE-EPCs react differently to CRP exposure and that at least part of the differences observed in the susceptibility to cell death may be linked to a lower percentage of progenitor cells expressing CD31 , for example, from patients with highest coronary plaque burden.

[0061] VEGF to EPC ratio is a novel biomarker of atherosclerosis in stable patients. Uniquely, quantitative information has been provided on the relationship between circulating progenitor cell number and coronary atheroma burden on IVUS in patients with stable CAD. The VEGF/EPC ratio correlated better with percent atheroma volume on IVUS and global plaque area score on QCA than did the absolute EPC count in blood or the serum VEGF concentration taken separately. Importantly, IVUS and QCA have independently provided confirmation of this observation. It is possible that the absence of an association between measures of plaque burden and VEGF/EPC ratio in unstable patients may result from a perturbation of normal homeostasis due to systemic inflammation, as reflected by increased hs-CRP concentrations and leukocyte numbers in these patients. The VEGF to EPC ratio provides further insights into the mechanisms of deficient vascular repair potential in CAD. It is believed that suboptimal VEGF-induced mobilization of EPCs is implicated in atherosclerosis progression. Possible mechanisms associated with this abnormality may include increased EPC removal (associated with an increased rate of EPC cell death) and/or deficiency of EPC production from the bone marrow. The ratio of serum VEGF to circulating EPC number therefore represents a circulating biomarker of atherosclerosis plaque burden and potentially, of vascular maintenance capacities in patients with stable CAD.

[0062] By way of analogy to the ratio between glucose and insulin, which can better describe insulin resistance than glucose and insulin levels taken separately, it is believed that a ratio of VEGF concentration to EPC number is indicative of inappropriate VEGF-induced EPC mobilization from the bone marrow to the circulation. Although VEGF is a critical factor in EPC mobilization, it can also enhance atherosclerosis progression through increased vascular permeability and inflammation, which seems opposite to the beneficial effects of EPCs. Consequently, the elevation in VEGF concentrations observed in stable CAD patients compared to in ACS patients, may reflect a compensatory response to atherosclerosis in stable CAD and be a factor for atherosclerosis progression. This response would also explain the strong correlation between the VEGF/EPC ratio and plaque burden in our study.

[0063] There was an apparent exhaustion of different hematopoietic lineages in stable patients. VEGF can have an effect on mobilization of EPCs but also on different hematopoietic lineages. Adenovector-mediated elevation of serum VEGF has been shown in mice to cause marked increases in total leukocyte counts, monocytes, colony-forming units of granulocytes-macrophages (CFU-GM), erythroid burst-forming units (BFU-E) and spleen colony-forming units (CFU-S) colony counts. In these experiments, the initial stimulation of hematopoiesis with increased bone marrow cellularity involving all lineages, was followed by reduced marrow cellularity by the third to fourth week. Observation of significant correlations between IVUS and QCA findings and both VEGF/leukocytes and VEGF/platelets ratio are consistent with the hypothesis that the production of the different hematopoietic cell lineages in response to VEGF stimulation is also deficient in stable patients with high coronary plaque burden.

[0064] HDL-cholesterol was observed as correlating with circulating CD34- positive cell and EPC numbers, as expected from recent findings in experimental models, in vitro experiments and after transplantation. In light of the inverse correlation between EPCs and plaque burden, it is suggested that the anti- atherosclerotic effects of HDL may be, at least partly, mediated by promotion of circulating numbers of EPC. The fact that HDL also correlated with the serum VEGF concentration also suggests that the relationship between HDL and EPCs may at least in part be mediated by VEGF-induced EPC mobilization. [0065] The early EPC CFU colony counts were found to correlate negatively with age. EPC CFU generation in culture is generally negatively related with the number of atherosclerosis risk factors in subjects without a history of CAD, but not in subjects with CAD. While variations in experimental methods probably account for some of these discrepancies, differences in clinical presentation, comorbidities such as left ventricular dysfunction, and concomitant medications may also be relevant. Whether EPC colony numbers in vitro are related to angiographic severity of coronary disease is controversial. Previous studies found no relationship between log-transformed endothelial cell CFU numbers and the number of diseased coronary arteries on angiography (see for example "Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353:999-1007."). In contrast, colony numbers were lower in patients with multivessel CAD compared with those with less severe disease or angiographically normal arteries in another study ("Kunz GA, Liang G, Cuculoski F, et al. Circulating endothelial progenitor cells predict coronary artery disease severity. Am Heart J 2006; 152:190-5."). Another study found that late-EPC colony numbers were actually higher in patients with most severe angiographic disease ("Guven H, Shepherd RM, Bach RG, Capoccia BJ, Link DC. The number of endothelial progenitor cell colonies in the blood is increased in patients with angiographically significant coronary artery disease. J Am Coll Cardiol 2006; 48:1579-87.8"). Different laboratory conditions including cell culture media, growth factors used and timing of analyses may have influenced the results of these studies. Given these uncertainties, our observations derived from peripheral patient blood present the VEGF/EPC ratio as an appealing alternative biomarker compared to CFU data from in vitro studies.

[0066] Conclusions

[0067] The susceptibility to cell death of early outgrowth culture-expanded EPCs from patients with CAD correlates with coronary plaque burden. In addition, the correlation between the VEGF/EPC ratio and coronary plaque burden indicates that insufficient or impaired vascular repair potential is linked with the severity of coronary atherosclerosis in stable patients. The VEGF/EPC ratio represents a novel circulating biomarker of vascular health and disease and the prognostic value of this marker and of culture-expanded EPC susceptibility to cell death. Therapeutic modification of circulating EPC number and function in patients with coronary heart disease may also be further explored.

[0068] Although the above embodiments have been described in language that is specific to certain structures, elements, compositions, and methodological steps, it is to be understood that the technology defined in the appended claims is not necessarily limited to the specific structures, elements, compositions and/or steps described. Rather, the specific aspects and steps are described as forms of implementing the claimed technology. Since many embodiments of the technology can be practiced without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended. Atty Docket No -21-

Table 1. Characteristics of the patients

All Stable CAD ACS^* p (stable

(n=54) (n=27) (n=27) vs. ACS)

Age, yrs 58 ±7 60 ±5 57 ±9 0.097

Men 46 (85) 20 (74) 26 (96) 0.022

Diabetes 7(13) 1 (4) 6 (22) 0.043

Hypertension 30 (56) 14 (52) 16 (59) 0.58

History of cigarette

25 (46) 18 (67) 7 (26) 0.003 smoking

History of chronic

35 (65) 22 (81) 13 (48) 0.010 angina

Previous myocardial

7 (13) 3 (11) 4 (15) 0.69 infarction

Previous stroke 4 (7) 3 (11) 1 (4) 0.30

ACE inhibitor or ARB 22 (41) 7 (26) 15 (56) 0.027

Beta-blocker 48 (89) 23 (85) 25 (93) 0.39

Statin 42 (78) 21 (78) 21 (78) 1.0

Systolic blood

127 ± 18 135 ± 18 119 ± 15 0.0008 pressure, mmHg

Diastolic blood

72±11 77 ± 11 67 ±8 0.0005 pressure, mmHg

Heart rate, beats per

64 ±9 63 ±7 64 ± 10 0.63 min

Creatinine, mmol/L 89 ±17 82 ± 13 95 ± 19 0.0035 hs-CRP, mg/L (n=49, 2.62 (1.0, 1.21 (0.6, 8.00 (2.2,

0.0001 26 and 23) 7.9) 3.1) 13.7) Atty Docket No -22-

Glucose, mmol/L 5.1 (4.6, 5.4 (4.9,

5.2 (4.8, 6.5) 0.030

(n=49, 26 and 23) 5.5) 7.8)

Serum VEGF (n=52,

252 ±213 312 ±244 191 ± 159 0.039 26 and 26)

Plasma lipids (n=49) (n=26) (n=23)

Total cholesterol,

4.5 ± 1.1 4.5 ±1.0 4.5 ± 1.1 0.87 mmol/L

LDL cholesterol, mmol/

2.4 ±0.9 2.4 ±0.8 2.4 ±1.0 0.85 L

HDL cholesterol,

1.2 ±0.3 1.3 ±0.2 1.1 ±0.3 0.088 mmol/L

Triglycerides, mmol/L 1.9 ±0.8 1.9±0.7 1.9 ±0.9 0.86

Total leukocyte count, x107L (n=50, 27 and 7.7 ±2.0 7.2 ±1.7 8.3 ±2.3 0.052

23)

Monocyte count, x10⁹/ 0.083 0.56

O 11

L (0.071, (0.44, <0.0001

(0.082, 0.59) (n=48, 26 and 22) 0.10) 0.81)

Endothelial progenitor 0.20

0.23 0.33 cells count, x10⁶/L (0.15, 0.12 (0.15, 0.37) (0.17, 0.48) (n=42, 20 and 22) 0.31)

IVUS findings

Percent atheroma 41.2 ±

39.8 ±9.8 38.5 ±9.5 0.33 volume, % 10.1

QCA findings

Cumulative coro.

2.5 ± 1.2 2.6 ± 1.3 2.4 ± 1.1 0.54 stenosis score Atty Docket No -23-

Global plaque area

38 ± 25 42 ± 30 34 ± 19 0.25 score, mm²

Coronary artery score,

2.2 ± 0.4 2.1 ± 0.4 2.2 ± 0.5 0.31 mm

^*Acute coronary syndromes (ACS): unstable angina, n= 15(56%); non-ST elevation myocardial infarction, n= 9(33%); ST-elevation myocardial infarction transferred < 7 days following index presentation for non-emergent coronary angiography, n= 3(1 1 %). Results are presented as mean±SD or n (%).

Atty Docket No -24-

Table 2. Correlations between VEGF/hematological counts ratio and IVUS or QCA measurements in stable patients

^*p<0.05

^*p<0.01

Claims

Atty. Docket No. -25-WHAT IS CLAIMED IS:

1. A method of detecting in a subject an indication associated with coronary artery disease, the method comprising: obtaining a biological sample comprising circulating endothelial progenitor cells (EPCs) and vascular endothelial growth factor (VEGF); measuring a ratio of a cell count of the EPCs and a concentration of the VEGF in the sample; and determining, based on the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample, whether the subject has the indication associated with coronary artery disease.

2. The method of claim 1 , wherein the biological sample is a blood sample.

3. The method of claim 2, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

4. The method of claim 1 , wherein the step of measuring the cell count of the EPCs is determined with flow cytometry.

5. The method of claim 1 , wherein the step of measuring the concentration of the VEGF is determined with ELISA.

6. A method of detecting a subject at risk of coronary artery disease, the method comprising: obtaining a biological sample comprising circulating endothelial progenitor cells (EPCs) and vascular endothelial growth factor (VEGF); measuring a ratio of a cell count of the EPCs and a concentration of the VEGF in the sample; and determining, based on the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample, the risk of coronary artery Atty Docket No -26 -

disease.

7. The method of claim 6, wherein the biological sample is a blood sample.

8. The method of claim 7, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

9. The method of claim 6, wherein the step of measuring the cell count of the EPCs is determined with flow cytometry.

10. The method of claim 6, wherein the step of measuring the concentration of the VEGF is determined with ELISA.

11. A method of detecting in a subject an indication associated with coronary artery disease, the method comprising: obtaining a biological sample from the subject comprising bone marrow- derived cells and a growth factor; measuring a ratio of a cell count of the bone marrow-derived cells and a concentration of the growth factor in the sample; and determining, based on the ratio of the bone marrow-derived cells and the concentration of the growth factor measured in the sample, whether the subject has the indication associated with coronary artery disease.

12. The method of claim 11 , wherein the biological sample is a blood sample.

13. The method of claim 12, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

14. The method of claim 11 , wherein the bone marrow-derived cells are circulating endothelial progenitor cells (EPCs). Atty Docket No. _2 7-

15. The method of claim 11 , wherein the bone marrow-derived cells are leucocytes.

16. The method of claim 11 , wherein the bone marrow-derived cells are platelets.

17. The method of claim 1 1 , wherein the step of measuring the cell count of the bone marrow-derived cells is determined with flow cytometry or an automated blood cell counter.

18. The method of claim 1 1 , wherein the growth factor is vascular endothelial growth factor (VEGF).

19. The method of claim 1 1 , wherein the step of measuring the concentration of the growth factor is determined with ELISA.

20. A method of detecting a subject at risk of coronary artery disease, the method comprising: obtaining a biological sample from the subject comprising bone marrow- derived cells and a growth factor; measuring a ratio of a cell count of the bone marrow-derived cells and a concentration of the growth factor in the sample; and determining, based on the ratio of the bone marrow-derived cells and the concentration of the growth factor measured in the sample, the risk of coronary artery disease.

21. The method of claim 20, wherein the biological sample is a blood sample.

22. The method of claim 21 , wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

23. The method of claim 20, wherein the bone marrow-derived cells are Atty Docket No -28- circulating endothelial progenitor cells (EPCs).

24. The method of claim 20, wherein the bone marrow-derived cells are leucocytes.

25. The method of claim 20, wherein the bone marrow-derived cells are platelets.

26. The method of claim 20, wherein the step of measuring the cell count of the bone marrow-derived cells is determined with flow cytometry or an automated blood cell counter.

27. The method of claim 20, wherein the growth factor is vascular endothelial growth factor (VEGF).

28. The method of claim 20, wherein the step of measuring the concentration of the growth factor is determined with ELISA.

29. A biomarker for detecting in a subject an indication associated with coronary artery disease, the biomarker comprising: a ratio of a cell count of circulating endothelial progenitor cells (EPCs) and a concentration of vascular endothelial growth factor (VEGF), measured in a biological sample comprising the EPCs and the VEGF, the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample, determining whether the subject has the indication associated with coronary artery disease.

30. The biomarker of claim 29, wherein the biological sample is a blood sample.

31 . The biomarker of claim 30, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease. Atty Docket No -29-

32. The biomarker of claim 29, wherein the cell count of the EPCs is determined with flow cytometry.

33. The biomarker of claim 29, wherein the concentration of the VEGF is determined with ELISA.

34. The biomarker for detecting a subject at risk of coronary artery disease, the biomarker comprising: a ratio of a cell count of circulating endothelial progenitor cells (EPCs) and a concentration of vascular endothelial growth factor (VEGF), measured in a biological sample comprising the EPCs and the VEGF, the ratio of the cell count of the EPCs and the concentration of the VEGF measured in the sample determining the risk of coronary artery disease.

35. The biomarker of claim 34, wherein the biological sample is a blood sample.

36. The biomarker of claim 35, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

37. The biomarker of claim 34, wherein the cell count of the EPCs is determined with flow cytometry.

38. The biomarker of claim 34, wherein the concentration of the VEGF is determined with ELISA.

39. A biomarker for detecting in a subject an indication associated with coronary artery disease, the biomarker comprising: a ratio of a cell count of bone marrow-derived cells and a concentration of a growth factor, measured in a biological sample comprising the bone marrow- Atty Docket No -30-

derived cells and the growth factor, the ratio of the cell count of the bone marrow- derived cells and the concentration of the growth factor measured in the sample, determining whether the subject has the indication associated with coronary artery disease.

40. The biomarker of claim 39, wherein the biological sample is a blood sample.

41. The biomarker of claim 40, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

42. The biomarker of claim 39, wherein the bone marrow-derived cells are circulating endothelial progenitor cells (EPCs).

43. The biomarker of claim 39, wherein the bone marrow-derived cells are leucocytes.

44. The biomarker of claim 39, wherein the bone marrow-derived cells are platelets.

45. The biomarker of claim 39, wherein the cell count of the bone marrow-derived cells is determined with flow cytometry or an automated blood cell counter.

46. The biomarker of claim 39, wherein the growth factor is vascular endothelial growth factor (VEGF).

47. The biomarker of claim 39, wherein the concentration of the growth factor is determined with ELISA.

48. A biomarker for detecting a subject at risk of coronary artery disease, the biomarker comprising: Atty. Docket No. -31 -

a ratio of a cell count of bone marrow-derived cells and a concentration of a growth factor, measured in a biological sample comprising the bone marrow- derived cells and the growth factor, the ratio of the cell count of the bone marrow- derived cells and the concentration of the growth factor measured in the sample determining the risk of coronary artery disease.

49. The biomarker of claim 48, wherein the biological sample is a blood sample.

50. The biomarker of claim 49, wherein the blood sample is removed from a location of the subject remote from plaque associated with the coronary artery disease.

51. The biomarker of claim 48, wherein the bone marrow-derived cells are circulating endothelial progenitor cells (EPCs).

52. The biomarker of claim 48, wherein the bone marrow-derived cells are leucocytes.

53. The biomarker of claim 48, wherein the bone marrow-derived cells are platelets.

54. The biomarker of claim 48, wherein the cell count of the bone marrow-derived cells is determined with flow cytometry or an automated blood cell counter.

55. The biomarker of claim 48, wherein the growth factor is vascular endothelial growth factor (VEGF).

56. The biomarker of claim 48, wherein the concentration of the growth factor is determined with ELISA.