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4(5): p. 1017-22.

1(7): p. 1575-9.

2005. 293(1): p. 90-5.

2003. 139(2): p. 137-47.

1033-41.

70(12): p. 2116-23.

*analysis.* Jama, 2005. 294(14): p. 1799-809.

*mortality?* Diabetes Care, 1996. 19(10): p. 1062-6.

Chronic Kidney Diseases: A New Insight and Emerging Strategies 249

[211] Danesh, J., S. Lewington, S.G. Thompson, et al., *Plasma fibrinogen level and the risk of*

[212] Marcucci, R., A.M. Gori, F. Giannotti, et al., *Markers of hypercoagulability and*

[213] Vicari, A.M., M.V. Taglietti, F. Pellegatta, et al., *Deranged platelet calcium homeostasis in*

[214] Haffner, S.M., L. Mykkanen, A. Festa, et al., *Insulin-resistant prediabetic subjects have more*

[216] Bloomgarden, Z.T., *Third Annual World Congress on the Insulin Resistance Syndrome:*

[217] Juhan-Vague, I., M.C. Alessi, A. Mavri, et al., *Plasminogen activator inhibitor-1,*

[218] Inomat, S., M. Haneda, T. Moriya, et al., *[Revised criteria for the early diagnosis of diabetic*

[219] Deferrari, G., M. Ravera, and V. Berruti, *Treatment of diabetic nephropathy in its early*

[220] Ferrier, K.E., M.H. Muhlmann, J.P. Baguest, et al., *Intensive Cholesterol Reduction lowers*

[221] Maiese, K., F. Li, and Z.Z. Chong, *New avenues of exploration for erythropoietin.* Jama,

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[224] Fox, K.M., *Efficacy of perindopril in reduction of cardiovascular events among patients with*

[225] *Randomised trial of a perindopril-based blood-pressure-lowering regimen among 6,105*

[226] Yusuf, S., P. Sleight, J. Pogue, et al., *Effects of an angiotensin-converting-enzyme inhibitor,*

[227] Bianchi, S., R. Bigazzi, and V.M. Campese, *Long-term effects of spironolactone on*

[228] Casas, J.P., W. Chua, S. Loukogeorgakis, et al., *Effect of inhibitors of the renin-angiotensin*

*Evaluation Study Investigators.* N Engl J Med, 2000. 342(3): p. 145-53.

*with the metabolic syndrome.* Diabetes Care, 2001. 24(4): p. 683-9.

*Atherothrombotic disease.* Diabetes Care, 2006. 29(8): p. 1973-80.

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*stages.* Diabetes Metab Res Rev, 2003. 19(2): p. 101-14.

American College of Cardiology, 2002. 39(6): p. 1020-5.

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**10**

*Italy*

*Cardiovascular Diseases Section,*

**Non Invasive Assessment of Cardiovascular**

Marco Matteo Ciccone\*, Michele Gesualdo, Annapaola Zito,

Cosimo Mandurino, Manuela Locorotondo and Pietro Scicchitano

**Risk Profile: The Role of the Ultrasound Markers**

*Department of Emergency and Organ Transplantation (DETO), University of Bari, Bari,*

Atherosclerosis, with its complications, is the most frequent cause of death all over the world, and it is the underlying cause of about 50% of all deaths in developed countries (1). Recent studies showed the key role played by inflammation and immune responses in development, progression, and rupture of atherosclerotic plaque (2,3,4). The presence of an immune reaction and/or infective antigens as potential triggers of atherogenesis (5,6) makes atherosclerosis be considered as an autoimmune disease in which the adaptive immune system is targeted against self-antigens modified by biochemical factors such as oxidative stress and hypercholesterolemia (7). These give rise to plaque birth (8,9) and the inflammatory status of the plaque makes the lesions unstable, inducing their abruption and acute thrombotic obstruction. Therefore, it induces impairment in endothelial function in bioactive antiatherogenic or proatherogenic molecules production (10), although other factors could increase such an imbalance: age (11), sex (12), hypertension (13), obesity (14), smoking (15), dyslipidemia (16), diabetes (17), all able to increase oxidative stress and vascular inflammation (18), morphological wall alterations and subsequently progression of

The initial atherosclerosis stages silently and symptom free occur since childhood (19); the clinical expressions (i.e., sudden cardiac death, myocardial infarction, angina pectoris, stroke, aortic aneurysm, renovascular hypertension, and intermittent claudication) involve 2 over 3 men and 1 over 2 women after age 40, and almost 60% of deaths are due to a cardiovascular disease cause (20). Thus, there has been an increase in recognition of the importance of subclinical atherosclerosis, and early detection of this insidious process must be the goal for improving cardiovascular health through prevention, and treatment of risk

Currently, non-invasive risk profile assessments can be evaluated not only with some laboratory parameters, (lipids and systemic inflammation markers as white blood cells,

**1. Introduction**

atheromatous lesions.

Corresponding Author

factors.

 \*

## **Non Invasive Assessment of Cardiovascular Risk Profile: The Role of the Ultrasound Markers**

Marco Matteo Ciccone\*, Michele Gesualdo, Annapaola Zito, Cosimo Mandurino, Manuela Locorotondo and Pietro Scicchitano *Cardiovascular Diseases Section, Department of Emergency and Organ Transplantation (DETO), University of Bari, Bari, Italy*

#### **1. Introduction**

250 Cardiovascular Risk Factors

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[230] Jafar, T.H., C.H. Schmid, M. Landa, et al., *Angiotensin-converting enzyme inhibitors and*

[231] Mann, J.F., W.M. McClellan, R. Kunz, et al., *Progression of renal disease--can we forget*

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[234] Bakris, G.L., P. Hart, and E. Ritz, *Beta blockers in the management of chronic kidney disease.*

[235] Khoshdel, A., S. Carney, A. Gillies, et al., *Potential roles of erythropoietin in the management of anaemia and other complications diabetes.* Diabetes Obes Metab, 2008. 10(1): p. 1-9. [236] Gross, J.L., M.J. de Azevedo, S.P. Silveiro, et al., *Diabetic nephropathy: diagnosis,*

[238] McGowan, T.A., Y. Zhu, and K. Sharma, *Transforming growth factor-beta: a clinical target for the treatment of diabetic nephropathy.* Curr Diab Rep, 2004. 4(6): p. 447-54. [239] Mangoni, A.A., R.A. Sherwood, B. Asonganyi, et al., *Short-term oral folic acid*

[240] Samuel, C.S. and T.D. Hewitson, *Relaxin in cardiovascular and renal disease.* Kidney Int,

[244] Lipton, S.A., *Erythropoietin for neurologic protection and diabetic neuropathy.* N Engl J Med,

[245] Gomez-Garre, D., J.L. Martin-Ventura, R. Granados, et al., *Losartan improves resistance*

[246] Oliver, J.J. and D.E. Newby, *Endothelial fibrinolytic function in hypertension: the expanding*

[247] Roldan, V. and F. Marin, *Are we content with lowering blood pressure alone, or should we be*

[249] Park, C.W., Y. Zhang, X. Zhang, et al., *PPARalpha agonist fenofibrate improves diabetic*

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*nephropathy in db/db mice.* Kidney Int, 2006. 69(9): p. 1511-7.

*artery lesions and prevents CTGF and TGF-beta production in mild hypertensive patients.*

*asking something more from the antihypertensive drugs we use?: effects of antihypertensive*

[241] Ashton, N., *Renal and vascular actions of urotensin II.* Kidney Int, 2006. 70(4): p. 624-9. [242] Varghese, Z., J.F. Moorhead, and X.Z. Ruan, *The PPARalpha ligand fenofibrate: meeting multiple targets in diabetic nephropathy.* Kidney Int, 2006. 69(9): p. 1490-1. [243] Vitale, C., G. Mercuro, A. Cornoldi, et al., *Metformin improves endothelial function in*

*patients with metabolic syndrome.* J Intern Med, 2005. 258(3): p. 250-6.

*supplementation enhances endothelial function in patients with type 2 diabetes.* Am J

*Consensus Meeting.* J Hypertens, 2006. 24(4): p. 603-10.

*prevention, and treatment.* Diabetes Care, 2005. 28(1): p. 164-76. [237] Bloomgarden, Z.T., *Diabetic nephropathy.* Diabetes Care, 2005. 28(3): p. 745-51.

*patient-level meta-analysis.* Ann Intern Med, 2003. 139(4): p. 244-52.

Med, 2001. 135(2): p. 73-87.

p. 2348-51; discussion 2352-3.

2004, www.medscape.com.

Kidney Int, 2006. 70(11): p. 1905-13.

Hypertens, 2005. 18(2 Pt 1): p. 220-6.

2006. 69(9): p. 1498-502.

2004. 350(24): p. 2516-7.

1673-4.

Kidney Int, 2006. 69(7): p. 1237-44.

*story.* J Hypertens, 2005. 23(8): p. 1471-2.

*blood pressure control, proteinuria, and angiotensin-converting enzyme inhibition: a*

*progression of nondiabetic renal disease. A meta-analysis of patient-level data.* Ann Intern

*about inhibition of the renin-angiotensin system?* Nephrol Dial Transplant, 2006. 21(9):

*patient with elevated heart rate: statement of a European Society of Hypertension*

Atherosclerosis, with its complications, is the most frequent cause of death all over the world, and it is the underlying cause of about 50% of all deaths in developed countries (1).

Recent studies showed the key role played by inflammation and immune responses in development, progression, and rupture of atherosclerotic plaque (2,3,4). The presence of an immune reaction and/or infective antigens as potential triggers of atherogenesis (5,6) makes atherosclerosis be considered as an autoimmune disease in which the adaptive immune system is targeted against self-antigens modified by biochemical factors such as oxidative stress and hypercholesterolemia (7). These give rise to plaque birth (8,9) and the inflammatory status of the plaque makes the lesions unstable, inducing their abruption and acute thrombotic obstruction. Therefore, it induces impairment in endothelial function in bioactive antiatherogenic or proatherogenic molecules production (10), although other factors could increase such an imbalance: age (11), sex (12), hypertension (13), obesity (14), smoking (15), dyslipidemia (16), diabetes (17), all able to increase oxidative stress and vascular inflammation (18), morphological wall alterations and subsequently progression of atheromatous lesions.

The initial atherosclerosis stages silently and symptom free occur since childhood (19); the clinical expressions (i.e., sudden cardiac death, myocardial infarction, angina pectoris, stroke, aortic aneurysm, renovascular hypertension, and intermittent claudication) involve 2 over 3 men and 1 over 2 women after age 40, and almost 60% of deaths are due to a cardiovascular disease cause (20). Thus, there has been an increase in recognition of the importance of subclinical atherosclerosis, and early detection of this insidious process must be the goal for improving cardiovascular health through prevention, and treatment of risk factors.

Currently, non-invasive risk profile assessments can be evaluated not only with some laboratory parameters, (lipids and systemic inflammation markers as white blood cells,

<sup>\*</sup> Corresponding Author

Non Invasive Assessment of Cardiovascular Risk Profile: The Role of the Ultrasound Markers 253

(40,41) through mechanical stimulation originating from increasing in vessel wall "shear stress". It is usually performed at brachial artery level by high-frequency ultrasonographic

It is performed in a quiet, temperature-controlled (22–24°C) room, early in the morning and it adopts a high resolution ultrasonograph connected to an image analysis system and a sphygmomanometer cuff applied around the forearm to create a flow stimulus in the brachial artery. The examination requires the patients to be supine, at rest, fast for at least 8 to 12 hours before the study; all vasoactive medications (calcium channel blockers, ßadrenergic blocking agents, nitrates and converting enzyme inhibitors) should be withheld for at least 4 half-lives, if possible. Moreover, subjects should avoid substances that might impair FMD such as caffeine, high-fat foods, and vitamin C or use tobacco for at least 4 to 6

**Hours** The examination should be performed at the same time of day **Temperature** Ultrasonographic evaluation should be performed at constant

**Drugs** All vasoactive drugs should be discontinued the night before

**Coffee and The** The day of the examination, the patient should refrain from

The 7,5 MHz electronic probe is positioned 4–5 cm above ante-cubital fossa to obtain longitudinal B-mode vascular scanning of the brachial artery with clear anterior and posterior intimal-lumen interfaces, and once the optimal artery image is achieved, the probe can be maintained in the right position using a mechanical arm. A pulsed wave Doppler

The procedure lasts 9 minutes: the first minute evaluate baseline diameter, measured at the

At the end of the first minute, the cuff is inflated 200-250 mmHg in order to close arterial inflow of the forearm (42). This causes ischemia and, consequentially, dilation of

After the sixth minute, the cuff is rapidly deflated: a brief high-flow state through the brachial artery to accommodate the dilated resistance vessels happens, and this reactive hyperemia produces a shear stress stimulus that induces the endothelium to release nitric oxide with subsequent vasodilation of the brachial artery between the 6th and 9th minute.

conditioning

taking coffee or tea

Table 1. Prerequisites and factors that influence the flow-mediated dilation

**Influence of food** Patients should not take copious meals or high in fat

the exam

**Smoking** Patients should abstain from smoking

**Brachial artery diameter** It must be between 2.5 and 5 mm

downstream resistance vessels by autoregulatory mechanisms.

temperature, in an environment equipped with air

imaging (21).

hours before the study (table 1).

**FACTORS COMMENTS**

recording is obtained from the midartery.

onset of the R-wave on the electrocardiogram.

reactive C protein and erythrocyte segmentation rate), but also with ultrasonographic methods that detect subclinical atherosclerosis. Three internationally validated methods had been adopted in order to evaluate endothelium function: brachial artery flow-mediated vasodilation (FMD) (21,22), antero-posterior abdominal aorta diameter (APAO) (23) and intima-media thickness of the common carotid artery (CCA-IMT) (24,25). Cause of their non-invasiveness, feasibility and cheap cost, these techniques are the best tools for the physicians to assess functional and morphological alterations of the arteries before a cardiovascular event occurs and the feasibility of therapies to reduce atherosclerosis burden (26).

### **2. FMD technique**

The endothelium is a real "organ", endowed with autocrine and paracrine properties and playing an essential role in controlling vasomotion by producing molecules able to modulate blood, such as nitric oxide (NO), the most important vasodilator molecule produced by endothelial cells) (27). Shear stress is the main element able to determine increasing in NO production, its action being exerted perpendicularly to the long axis of the vessel.

Nevertheless, endothelial cells can also produce substances with vasoconstrictor action, as endothelin-1, (27) above all in case of increased age, hyperhomocysteinemia, smoking, diabetes, hypercholesterolemia, and hypertension (28): in these case it could be detected the presence of reduced vasodilating response to endothelial stimuli. Instead, diet and exercise can improve endothelial function (29). Lipid-lowering therapy (30,31), antioxidants (32), estrogen replacement (33) and treatment with angiotensin-converting enzyme inhibition or receptor blockade (34) improve this response.

Thus, endothelial dysfunction is considered the basic pathogenic mechanism of cardiovascular disease (35) and therefore can be considered as an early marker of cardiovascular risk.

In fact, the endothelial dysfunction seems to be the earliest event in the process of atherosclerotic plaque formation, appearing even before structural lesion of the vessel wall (36); for this reason the evaluation of endothelial function could be a useful tool for early stratification of patients at risk for cardiovascular events.

Studies in postmenopausal women suggest that endothelial dysfunction may be a predisposing factor for the development of hypertension (37) and diabetes (38), thus being not only a consequence of risk factors but also a pathogenetic mechanism for their onset.

Moreover, impaired endothelium-dependent vasomotion may contribute to the genesis of cardiovascular events by modulating the stability of plaque and coronary vasospasm. In fact, the analysis of Lerman and Zeiher (39) showed that endothelial dysfunction, assessed both at coronary and peripheral level, is significantly predictive of cardiovascular events independently of the presence of traditional cardiovascular risk factors.

#### **3. Procedure description**

A non-invasive method to assess endothelium-dependent flow-mediated vasodilation (FMD) was developed in the 1990s: it consisted in inducing endothelial cells to release NO

reactive C protein and erythrocyte segmentation rate), but also with ultrasonographic methods that detect subclinical atherosclerosis. Three internationally validated methods had been adopted in order to evaluate endothelium function: brachial artery flow-mediated vasodilation (FMD) (21,22), antero-posterior abdominal aorta diameter (APAO) (23) and intima-media thickness of the common carotid artery (CCA-IMT) (24,25). Cause of their non-invasiveness, feasibility and cheap cost, these techniques are the best tools for the physicians to assess functional and morphological alterations of the arteries before a cardiovascular event occurs and the feasibility of therapies to reduce atherosclerosis burden

The endothelium is a real "organ", endowed with autocrine and paracrine properties and playing an essential role in controlling vasomotion by producing molecules able to modulate blood, such as nitric oxide (NO), the most important vasodilator molecule produced by endothelial cells) (27). Shear stress is the main element able to determine increasing in NO

Nevertheless, endothelial cells can also produce substances with vasoconstrictor action, as endothelin-1, (27) above all in case of increased age, hyperhomocysteinemia, smoking, diabetes, hypercholesterolemia, and hypertension (28): in these case it could be detected the presence of reduced vasodilating response to endothelial stimuli. Instead, diet and exercise can improve endothelial function (29). Lipid-lowering therapy (30,31), antioxidants (32), estrogen replacement (33) and treatment with angiotensin-converting enzyme inhibition or

Thus, endothelial dysfunction is considered the basic pathogenic mechanism of cardiovascular disease (35) and therefore can be considered as an early marker of

In fact, the endothelial dysfunction seems to be the earliest event in the process of atherosclerotic plaque formation, appearing even before structural lesion of the vessel wall (36); for this reason the evaluation of endothelial function could be a useful tool for early

Studies in postmenopausal women suggest that endothelial dysfunction may be a predisposing factor for the development of hypertension (37) and diabetes (38), thus being not only a consequence of risk factors but also a pathogenetic mechanism for their onset.

Moreover, impaired endothelium-dependent vasomotion may contribute to the genesis of cardiovascular events by modulating the stability of plaque and coronary vasospasm. In fact, the analysis of Lerman and Zeiher (39) showed that endothelial dysfunction, assessed both at coronary and peripheral level, is significantly predictive of cardiovascular events

A non-invasive method to assess endothelium-dependent flow-mediated vasodilation (FMD) was developed in the 1990s: it consisted in inducing endothelial cells to release NO

production, its action being exerted perpendicularly to the long axis of the vessel.

(26).

**2. FMD technique**

cardiovascular risk.

**3. Procedure description**

receptor blockade (34) improve this response.

stratification of patients at risk for cardiovascular events.

independently of the presence of traditional cardiovascular risk factors.

(40,41) through mechanical stimulation originating from increasing in vessel wall "shear stress". It is usually performed at brachial artery level by high-frequency ultrasonographic imaging (21).

It is performed in a quiet, temperature-controlled (22–24°C) room, early in the morning and it adopts a high resolution ultrasonograph connected to an image analysis system and a sphygmomanometer cuff applied around the forearm to create a flow stimulus in the brachial artery. The examination requires the patients to be supine, at rest, fast for at least 8 to 12 hours before the study; all vasoactive medications (calcium channel blockers, ßadrenergic blocking agents, nitrates and converting enzyme inhibitors) should be withheld for at least 4 half-lives, if possible. Moreover, subjects should avoid substances that might impair FMD such as caffeine, high-fat foods, and vitamin C or use tobacco for at least 4 to 6 hours before the study (table 1).

Table 1. Prerequisites and factors that influence the flow-mediated dilation

The 7,5 MHz electronic probe is positioned 4–5 cm above ante-cubital fossa to obtain longitudinal B-mode vascular scanning of the brachial artery with clear anterior and posterior intimal-lumen interfaces, and once the optimal artery image is achieved, the probe can be maintained in the right position using a mechanical arm. A pulsed wave Doppler recording is obtained from the midartery.

The procedure lasts 9 minutes: the first minute evaluate baseline diameter, measured at the onset of the R-wave on the electrocardiogram.

At the end of the first minute, the cuff is inflated 200-250 mmHg in order to close arterial inflow of the forearm (42). This causes ischemia and, consequentially, dilation of downstream resistance vessels by autoregulatory mechanisms.

After the sixth minute, the cuff is rapidly deflated: a brief high-flow state through the brachial artery to accommodate the dilated resistance vessels happens, and this reactive hyperemia produces a shear stress stimulus that induces the endothelium to release nitric oxide with subsequent vasodilation of the brachial artery between the 6th and 9th minute.

Non Invasive Assessment of Cardiovascular Risk Profile: The Role of the Ultrasound Markers 255

Intima-media thickness of abdominal aorta has been firstly suggested as cardiovascular risk

Recently, in addition to arterial wall thickening, attention has been paid on APAO as a possible early marker of atherosclerosis (before clinical manifestations have become evident). Indeed, arterial dilatation is a well-known age-related manifestation, and some of the molecular events causing this alterations are involved in the pathogenesis of

There is a relationship between APAO in the non-aneurysmal range (<30 mm in diameter) and all-cause mortality: in a cohort of 12203 men aged 65 years and older infrarenal aortic diameter is turned out to be an independent predictor of all-cause mortality, particularly cardiovascular mortality (52). In another study on 4734 participants > 65 years old underwent to abdominal aortic ultrasound evaluation, has been demonstrated that for those with an infrarenal aortic diameters >2.0 cm, there was a significantly higher risk of future cardiovascular events and total mortality, suggesting a value of infrarenal aortic diameters

Furtheremore, Allison et al (54) showed that age, gender, body mass index, and the presence and extent of calcified atherosclerosis in both the abdominal aorta and iliac arteries are significantly associated with increasing aortic diameter independently of other cardiovascular risk factors. A study by Ciccone et al. involving women with polycystic ovary syndrome PCOS (55) showed that the increase in APAO is the earliest arterial alteration in women with PCOS, thus preceding the IMT of other arteries such as common carotid arteries and common femoral

However, this alteration seems to be due to body weight secondary to PCOS and not to PCOS *per se*. In fact, Gorter PM et al (56) showed that intra-abdominal fat accumulation and metabolic syndrome are associated with larger infrarenal aortic diameter in patients with clinically evident arterial disease, indicating a role for intra-abdominal fat in the

To explain these findings it can be hypothesized that APAO may represent a measure of cumulative exposure to genetic and environmental risk factors implicated in atherosclerosis development. For these reasons, APAO can be considered as an early marker of cardiovascular risk, and because of its noninvasive measurement and feasibility might be used to investigate determinants of atherosclerosis at an early stage of the process and to assess modifiers of atherosclerosis disease progression, such as lifestyle and

Wilmink and colleagues (57,58) showed that the use of ultrasounds to measure the infrarenal aortic diameter is attractive as it is rapid, cheap, and noninvasive. The good accuracy of infrarenal aortic diameter measurements by ultrasound makes this method

With the patient in supine position, the examination is carried out with a 3.5 MHz electronic probe placed one centimetre left of the umbilicus. The longitudinal ultrasound scans allow the

between 2.0 and 3.0 cm as another manifestation of subclinical atherosclerosis (53).

arteries. This identifies APAO as an early marker of atherosclerosis.

development of larger aortic diameters.

pharmacological interventions.

**5. Procedure description**

acceptable for clinical decision-making.

marker in patients stratification risk profile (49).

cardiovascular disease (50,51).

The software calculates FMD value as percentage of increasing of diameter value from baseline:

FMD = [(postiperemia diameter - baseline diameter) / baseline diameter] x 100

The maximal increase in diameter occurs approximately 60 to 90 seconds after cuff release. FMD values greater than 5-10% are considered "normal" (21). A schematic overview of this imaging technique could be observed in figure 1a.

This reactive hyperemia phase is confirmed by measuring the arterial blood flow using pulse-wave Doppler. The peak blood flow in the brachial artery is obtained with the sample volume in the centre of the artery and a correction angle of 70°. It is estimated at rest and during the first 15 s after cuff deflation, taking the average of the pulsed Doppler velocity signal of 3 measurements. The maximum speeds considered normal is 50–70 cm/s. Reactive hyperemia is calculated as the ratio of the maximal velocity divided by the maximal velocity at baseline.

Because of its low reproducibility and accuracy (43,44), the technique requires very high methodology accuracy and a mechanical support for the probe with micrometer adjustment to prevent movement of the vascular probe, and specific software ("FMD Studio") to measure second to second changes in artery caliber (21). The variations in caliber measured are small (from 0 to 15%), so the FMD represents a stimulus-type "on / off" poorly modulated.

Therefore, in order to obtain results that have a clinical validity, it is necessary to study a large number of patients. In support of the role of endothelial function as marker of cardiovascular risk and of the validity of the FMD method, there is also correlation with the invasive test data of coronary endothelial function (45) and with the severity and extent of atherosclerosis coronary (46).

Moreover, the noninvasive nature of the technique allows repeated measurements over time to study the effectiveness of various interventions that may affect vascular health.

#### **4. APAO**

Up to now the infrarenal anteroposterior diameter of abdominal aorta (APAO) has been always related to the abdominal aortic aneurysms (AAAs), as a measurement to be used in the diagnostic and follow-up phase of this disease and for surgical intervention planning.

An abdominal aortic aneurysm is defined by some authors as an infrarenal aortic diameter ≥ 3.0 cm, or a ratio between infrarenal and suprarenal aorta diameters greater than 1.2, all measured by ultrasound B-mode (47). As coronary heart disease and stroke continue to be the leading causes of death and disability among adults in developed countries, an early detection of vascular damage and, consequently, adequate cardiovascular risk stratification has received an intense attention in the last years in order to decrease the impact of cardiovascular disease.

To detect the "primum movens" of atherosclerotic disease, several studies have been conducted in the last years for identify new ultrasonographic markers (48).

Intima-media thickness of abdominal aorta has been firstly suggested as cardiovascular risk marker in patients stratification risk profile (49).

Recently, in addition to arterial wall thickening, attention has been paid on APAO as a possible early marker of atherosclerosis (before clinical manifestations have become evident). Indeed, arterial dilatation is a well-known age-related manifestation, and some of the molecular events causing this alterations are involved in the pathogenesis of cardiovascular disease (50,51).

There is a relationship between APAO in the non-aneurysmal range (<30 mm in diameter) and all-cause mortality: in a cohort of 12203 men aged 65 years and older infrarenal aortic diameter is turned out to be an independent predictor of all-cause mortality, particularly cardiovascular mortality (52). In another study on 4734 participants > 65 years old underwent to abdominal aortic ultrasound evaluation, has been demonstrated that for those with an infrarenal aortic diameters >2.0 cm, there was a significantly higher risk of future cardiovascular events and total mortality, suggesting a value of infrarenal aortic diameters between 2.0 and 3.0 cm as another manifestation of subclinical atherosclerosis (53).

Furtheremore, Allison et al (54) showed that age, gender, body mass index, and the presence and extent of calcified atherosclerosis in both the abdominal aorta and iliac arteries are significantly associated with increasing aortic diameter independently of other cardiovascular risk factors. A study by Ciccone et al. involving women with polycystic ovary syndrome PCOS (55) showed that the increase in APAO is the earliest arterial alteration in women with PCOS, thus preceding the IMT of other arteries such as common carotid arteries and common femoral arteries. This identifies APAO as an early marker of atherosclerosis.

However, this alteration seems to be due to body weight secondary to PCOS and not to PCOS *per se*. In fact, Gorter PM et al (56) showed that intra-abdominal fat accumulation and metabolic syndrome are associated with larger infrarenal aortic diameter in patients with clinically evident arterial disease, indicating a role for intra-abdominal fat in the development of larger aortic diameters.

To explain these findings it can be hypothesized that APAO may represent a measure of cumulative exposure to genetic and environmental risk factors implicated in atherosclerosis development. For these reasons, APAO can be considered as an early marker of cardiovascular risk, and because of its noninvasive measurement and feasibility might be used to investigate determinants of atherosclerosis at an early stage of the process and to assess modifiers of atherosclerosis disease progression, such as lifestyle and pharmacological interventions.

#### **5. Procedure description**

254 Cardiovascular Risk Factors

The software calculates FMD value as percentage of increasing of diameter value from

FMD = [(postiperemia diameter - baseline diameter) / baseline diameter] x 100 The maximal increase in diameter occurs approximately 60 to 90 seconds after cuff release. FMD values greater than 5-10% are considered "normal" (21). A schematic overview of this

This reactive hyperemia phase is confirmed by measuring the arterial blood flow using pulse-wave Doppler. The peak blood flow in the brachial artery is obtained with the sample volume in the centre of the artery and a correction angle of 70°. It is estimated at rest and during the first 15 s after cuff deflation, taking the average of the pulsed Doppler velocity signal of 3 measurements. The maximum speeds considered normal is 50–70 cm/s. Reactive hyperemia is calculated as the ratio of the maximal velocity divided by the maximal velocity

Because of its low reproducibility and accuracy (43,44), the technique requires very high methodology accuracy and a mechanical support for the probe with micrometer adjustment to prevent movement of the vascular probe, and specific software ("FMD Studio") to measure second to second changes in artery caliber (21). The variations in caliber measured are small (from 0 to 15%), so the FMD represents a stimulus-type "on / off" poorly

Therefore, in order to obtain results that have a clinical validity, it is necessary to study a large number of patients. In support of the role of endothelial function as marker of cardiovascular risk and of the validity of the FMD method, there is also correlation with the invasive test data of coronary endothelial function (45) and with the severity and extent of

Moreover, the noninvasive nature of the technique allows repeated measurements over time

Up to now the infrarenal anteroposterior diameter of abdominal aorta (APAO) has been always related to the abdominal aortic aneurysms (AAAs), as a measurement to be used in the diagnostic and follow-up phase of this disease and for surgical intervention planning.

An abdominal aortic aneurysm is defined by some authors as an infrarenal aortic diameter ≥ 3.0 cm, or a ratio between infrarenal and suprarenal aorta diameters greater than 1.2, all measured by ultrasound B-mode (47). As coronary heart disease and stroke continue to be the leading causes of death and disability among adults in developed countries, an early detection of vascular damage and, consequently, adequate cardiovascular risk stratification has received an intense attention in the last years in order to decrease the impact of

To detect the "primum movens" of atherosclerotic disease, several studies have been

conducted in the last years for identify new ultrasonographic markers (48).

to study the effectiveness of various interventions that may affect vascular health.

imaging technique could be observed in figure 1a.

baseline:

at baseline.

modulated.

**4. APAO**

atherosclerosis coronary (46).

cardiovascular disease.

Wilmink and colleagues (57,58) showed that the use of ultrasounds to measure the infrarenal aortic diameter is attractive as it is rapid, cheap, and noninvasive. The good accuracy of infrarenal aortic diameter measurements by ultrasound makes this method acceptable for clinical decision-making.

With the patient in supine position, the examination is carried out with a 3.5 MHz electronic probe placed one centimetre left of the umbilicus. The longitudinal ultrasound scans allow the

Non Invasive Assessment of Cardiovascular Risk Profile: The Role of the Ultrasound Markers 257

measurements are more accurate for the distal than for the proximal aorta measurement and the definition of the aortic diameter based on a combination of both distal and proximal

Atherosclerosis is a disease with a slowly progressive course and a long asymptomatic period. The clinical manifestations generally appear in middle age (63), and the first event

Since the atherosclerotic disease is a multidistrict and multifocal process, identifying the changes of the vascular wall at subclinical stages of atherosclerosis is essential in assessing global cardiovascular risk (64) and in promoting the use of preventive strategies, as well as

Among imaging techniques for detection of early preclinical stages of atherosclerosis, the best is the measurement of the carotid intima-media thickness (CCA-IMT) using ultrasound high-resolution B-mode; the evaluation of this parameter is a noninvasive and reproducible

It is a well-validated research tool that has been translated increasingly into clinical practice

Many studies demonstrated the role of CCA-IMT in the early evaluation of atherosclerosis disease. In fact, this parameter was found to be associated with the presence of cardiovascular risk factors (67,68,69) and with atherosclerotic lesions in other vascular districts, such as coronary and lower extremity arteries (70,71,72). Gasparyan (73) already put on evidence the importance of carotid ultrasound assessment in the clinical practice. Apart from CCA-IMT evaluation, the ultrasound evaluation should consider all the characteristics of carotid wall: it is necessary to evaluate IMT and, at the same time,

Prospective epidemiological studies showed that individuals with elevated carotid IMT are more likely to suffer from cardiovascular or cerebrovascular events, suggesting that thickened carotid IMT is a powerful and independent indicator of the likelihood of general arteriosclerosis (74,75). The predictive power of carotid IMT is maintained even after adjustment for major cardiovascular risk factors. Thus, measurement of IMT may provide informations in addition to traditional risk factors during assessment of global

SSeveral works in the last decade confirmed the role of this parameter in the early detection

Moreover, changes in carotid IMT may be used as a measure of efficacy of pharmacologic

Carotid ultrasound can be performed using vascular echographic apparatus equipped with high-frequency transducers (usually 3-10 MHz and linear array) and appropriate software.

measurement may be more accurate.

triggered by atherosclerosis can be fatal.

as a cardiovascular risk marker (65,66).

morphological aspects of carotid wall.

intervention.

**7. Procedure description**

cardiovascular risk profile in asymptomatic subjects (76).

of atherosclerosis and in measure of its severity (77,78).

optimization of preventive and protective care.

method for identifying and quantifying subclinical vascular disease.

**6. IMT**

study of the aorta and the best image in long axis projection of the abdominal aorta is used for the measurement. To improve the image acquisition, subjects are asked to keep fasting for at least 6-8 hours and follow a fiber diet for the two days prior to the examination to reduce intestinal bloating (diet preparation). To reduce the bias and interobserver variability the study of infrarenal abdominal aorta should be performed by same physician (59,60).

In the study of Ciccone et al.(55) the anteroposterior diameter of the aorta was defined as the maximal external cross-sectional measurement. It was calculated as the distance between the near and the far walls of the abdominal aorta on images that were frozen in systole. All the measurements were performed at 0.5, 1, and 2 cm above the umbilicus and were expressed in centimetres (see also Figure 1c and 1d).

Fig. 1. a) Flow-mediated vasodilation (FMD) technique image. **b)** Ultrasound evaluation of common carotid artery intima-media thickness. **c)** Antero-posterior abdominal aorta diameter in long axis views. **d)** Antero-posterior abdominal aorta diameter in short axis views

However, in several studies the position of the probe and the part of abdominal aorta evaluated may be different.

van den Bosch et al. (61) studied distal aortic diameter to assess the relationship between abdominal aortic diameter and peripheral arterial occlusive disease. They demonstrated that both patients with an aortic diameter too large and patients with an aortic diameter too small are prone to peripheral arterial occlusive disease.

The study of Norman P. et al. (52) was carried out using a 3.75 mol/L Hz probe to measure the maximum transverse and antero-posterior diameter of the infrarenal aorta. The largest measurement was recorded as the aortic diameter.

Pleumeekers et al. (62) evaluated the observer variability of ultrasound measurements of proximal and distal part of the abdominal aorta. Their results were that ultrasound measurements are more accurate for the distal than for the proximal aorta measurement and the definition of the aortic diameter based on a combination of both distal and proximal measurement may be more accurate.

### **6. IMT**

256 Cardiovascular Risk Factors

study of the aorta and the best image in long axis projection of the abdominal aorta is used for the measurement. To improve the image acquisition, subjects are asked to keep fasting for at least 6-8 hours and follow a fiber diet for the two days prior to the examination to reduce intestinal bloating (diet preparation). To reduce the bias and interobserver variability the study

In the study of Ciccone et al.(55) the anteroposterior diameter of the aorta was defined as the maximal external cross-sectional measurement. It was calculated as the distance between the near and the far walls of the abdominal aorta on images that were frozen in systole. All the measurements were performed at 0.5, 1, and 2 cm above the umbilicus and were expressed

Fig. 1. a) Flow-mediated vasodilation (FMD) technique image. **b)** Ultrasound evaluation of common carotid artery intima-media thickness. **c)** Antero-posterior abdominal aorta diameter in long axis views. **d)** Antero-posterior abdominal aorta diameter in short axis

However, in several studies the position of the probe and the part of abdominal aorta

van den Bosch et al. (61) studied distal aortic diameter to assess the relationship between abdominal aortic diameter and peripheral arterial occlusive disease. They demonstrated that both patients with an aortic diameter too large and patients with an aortic diameter too

The study of Norman P. et al. (52) was carried out using a 3.75 mol/L Hz probe to measure the maximum transverse and antero-posterior diameter of the infrarenal aorta. The largest

Pleumeekers et al. (62) evaluated the observer variability of ultrasound measurements of proximal and distal part of the abdominal aorta. Their results were that ultrasound

of infrarenal abdominal aorta should be performed by same physician (59,60).

in centimetres (see also Figure 1c and 1d).

views

evaluated may be different.

small are prone to peripheral arterial occlusive disease.

measurement was recorded as the aortic diameter.

Atherosclerosis is a disease with a slowly progressive course and a long asymptomatic period. The clinical manifestations generally appear in middle age (63), and the first event triggered by atherosclerosis can be fatal.

Since the atherosclerotic disease is a multidistrict and multifocal process, identifying the changes of the vascular wall at subclinical stages of atherosclerosis is essential in assessing global cardiovascular risk (64) and in promoting the use of preventive strategies, as well as optimization of preventive and protective care.

Among imaging techniques for detection of early preclinical stages of atherosclerosis, the best is the measurement of the carotid intima-media thickness (CCA-IMT) using ultrasound high-resolution B-mode; the evaluation of this parameter is a noninvasive and reproducible method for identifying and quantifying subclinical vascular disease.

It is a well-validated research tool that has been translated increasingly into clinical practice as a cardiovascular risk marker (65,66).

Many studies demonstrated the role of CCA-IMT in the early evaluation of atherosclerosis disease. In fact, this parameter was found to be associated with the presence of cardiovascular risk factors (67,68,69) and with atherosclerotic lesions in other vascular districts, such as coronary and lower extremity arteries (70,71,72). Gasparyan (73) already put on evidence the importance of carotid ultrasound assessment in the clinical practice. Apart from CCA-IMT evaluation, the ultrasound evaluation should consider all the characteristics of carotid wall: it is necessary to evaluate IMT and, at the same time, morphological aspects of carotid wall.

Prospective epidemiological studies showed that individuals with elevated carotid IMT are more likely to suffer from cardiovascular or cerebrovascular events, suggesting that thickened carotid IMT is a powerful and independent indicator of the likelihood of general arteriosclerosis (74,75). The predictive power of carotid IMT is maintained even after adjustment for major cardiovascular risk factors. Thus, measurement of IMT may provide informations in addition to traditional risk factors during assessment of global cardiovascular risk profile in asymptomatic subjects (76).

SSeveral works in the last decade confirmed the role of this parameter in the early detection of atherosclerosis and in measure of its severity (77,78).

Moreover, changes in carotid IMT may be used as a measure of efficacy of pharmacologic intervention.

#### **7. Procedure description**

Carotid ultrasound can be performed using vascular echographic apparatus equipped with high-frequency transducers (usually 3-10 MHz and linear array) and appropriate software.

Non Invasive Assessment of Cardiovascular Risk Profile: The Role of the Ultrasound Markers 259

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The patient should be positioned supine with slight (45°) hyperextension and rotation of the neck in the direction opposite the probe.

CCA-IMT is defined as the distance between the lumen-intima interface and the mediaadventitia interface, which corresponds to the inner and outer echogenic lines seen on the Bmode ultrasound image [see figure 1b] (24,79).

Measurement of carotid IMT (c-IMT) is traditionally performed with the image of the carotid artery in the longitudinal axis, revealing the common carotid artery, the carotid bifurcation, and the internal and external carotid arteries.

Although these measurements have been performed for years, significant variability exists when measuring the near wall due to technical and acoustic difficulties encountered when imaging the c-IMT of the near wall (80). Due to these technical limitations, clinical measurement of c-IMT using B-mode ultrasound is often applied to the far (posterior) wall of the common carotid artery.

IMT is measured at about 2 cm proximal to the dilation of the bulb of the common carotid artery.

Three measurements coming from three different sites [according to the method described by Pignoli et al. (79): about 2 cm above the flow-divider, about ½ cm above the flow-divider and in middle zone] are considered for IMT evaluation. An average of all these values would be calculated at the end of the measures.

Mean IMT (m-IMT) and maximum IMT (M-IMT) are measured. m-IMT represents the mean value of all measurements at each common carotid artery, averaging the left and right sides. M-IMT represents the mean value of the single highest IMT measurements at each common carotid artery, averaging the left and right sides. Carotid plaque is defined as the presence of a greater than 1.5 mm c-IMT measurement or an area within the carotid artery that is at least 50% greater than the size of the surrounding vessel wall.

The same physician should perform the evaluation in order to reduce bias and improve the results.

A problem associated with the ultrasonographic IMT measurement is the variation in the readings, which leads to different results of repeated measurements from the same observer. In general, the inter- and intra-observer errors are acceptable and the technique has a good reproducibility (81,82).

#### **8. References**

The patient should be positioned supine with slight (45°) hyperextension and rotation of the

CCA-IMT is defined as the distance between the lumen-intima interface and the mediaadventitia interface, which corresponds to the inner and outer echogenic lines seen on the B-

Measurement of carotid IMT (c-IMT) is traditionally performed with the image of the carotid artery in the longitudinal axis, revealing the common carotid artery, the carotid

Although these measurements have been performed for years, significant variability exists when measuring the near wall due to technical and acoustic difficulties encountered when imaging the c-IMT of the near wall (80). Due to these technical limitations, clinical measurement of c-IMT using B-mode ultrasound is often applied to the far (posterior) wall

IMT is measured at about 2 cm proximal to the dilation of the bulb of the common carotid

Three measurements coming from three different sites [according to the method described by Pignoli et al. (79): about 2 cm above the flow-divider, about ½ cm above the flow-divider and in middle zone] are considered for IMT evaluation. An average of all these values

Mean IMT (m-IMT) and maximum IMT (M-IMT) are measured. m-IMT represents the mean value of all measurements at each common carotid artery, averaging the left and right sides. M-IMT represents the mean value of the single highest IMT measurements at each common carotid artery, averaging the left and right sides. Carotid plaque is defined as the presence of a greater than 1.5 mm c-IMT measurement or an area within the carotid artery that is at least

The same physician should perform the evaluation in order to reduce bias and improve the

A problem associated with the ultrasonographic IMT measurement is the variation in the readings, which leads to different results of repeated measurements from the same observer. In general, the inter- and intra-observer errors are acceptable and the technique has a good

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**11**

*1Australia 2USA*

**Endothelial Progenitor Cell Number:**

Michel R. Hoenig1 and Frank W. Sellke2

*2Department of Cardiothoracic Surgery,*

*1Department of Surgery, The Alfred Hospital, Melbourne*

*Rhode Island Hospital and Brown Medical School, Providence,*

**A Convergence of Cardiovascular Risk Factors**

The bone marrow of adult humans is a source of endothelial progenitor cells (EPCs) that circulate in the blood and repair damaged endothelium. The number and function of EPCs is predictive of endothelial function and cardiovascular events. Herein we discuss the impact of individual risk factors on EPC numbers and discuss the potential utility of EPC number as a cardiovascular risk-assessment tool that integrates traditional and emerging

Cardiovascular disease the leading cause of mortality in the Western world and manifests as coronary disease, peripheral vascular disease, or ischemic stroke depending on the vascular territory affected. The ageing population and projected increases in prevalence and costs of care have highlighted the need for more effective prevention of cardiovascular disease (Heidenreich, Trogdon et al. 2011). These manifestations of cardiovascular disease share common risk factors of age, hypertension, diabetes, hypercholesterolemia and smoking (Roger, Go et al. 2011). Endothelial dysfunction is the precursor lesion to atherosclerosis and reflects depressed nitric oxide (NO) release from the endothelium (Furchgott 1996; Valgimigli, Merli et al. 2003). Basal release of NO from the endothelium regulates vascular tone and antagonizes the actions of vasoconstrictor substances. Further, NO possesses antiplatelet actions and down-regulates adhesion molecules that attract inflammatory cells to the endothelium (Deanfield, Halcox et al. 2007). The degree of endothelial dysfunction shows a graded response to the number of cardiac risk factors present (Bonetti, Lerman et al. 2003; Davignon and Ganz 2004) and is predictive of clinical events (Bonetti, Lerman et al. 2003; Davignon and Ganz 2004; Deanfield, Halcox et al. 2007). Since endothelial dysfunction occurs systemically, the atherosclerotic process involves a large portion of the arterial tree before it becomes clinically manifest (Deanfield, Halcox et al. 2007). Patient presentations to a cardiologist, cardiac surgeon, vascular surgeon or stroke neurologist with clinically manifest atherosclerosis are typically preceded by decades of endothelial dysfunction and depressed vascular repair throughout the entire arterial bed (Ross 1993). The systemic

**1. Introduction**

cardiovascular risk factors.

**2. The systemic basis of cardiovascular disease**

## **Endothelial Progenitor Cell Number: A Convergence of Cardiovascular Risk Factors**

Michel R. Hoenig1 and Frank W. Sellke2

*1Department of Surgery, The Alfred Hospital, Melbourne 2Department of Cardiothoracic Surgery, Rhode Island Hospital and Brown Medical School, Providence, 1Australia 2USA*

#### **1. Introduction**

264 Cardiovascular Risk Factors

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Reproducibility in the PARC Study. *Cerebrovascular Diseases.* 19:57-63, 2005.

Study on Atherosclerosis (ELSA). *J. Hypertens* 2000;18:197-2018.

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secondary prevention. Identifying the high-risk patient for primary prevention: noninvasive tests of atherosclerotic burden. Writing Group III. *Circulation* 2000; 101:

Konieczynska M. Association of increased carotid intima-media thickness with the

intimamedia thickness at different sites: relation to incident myocardial infarction;

arterial wall: a direct measurement with ultrasound imaging. *Circulation.*

walls of carotid arteries using B-mode ultrasound. *Ultrasound Med Biol.* 1996;

measurement of carotid artery intima-media thickness: the European Lacidipine

Merli Design, Baseline Characteristics and Carotid Intima-Media Thickness

The bone marrow of adult humans is a source of endothelial progenitor cells (EPCs) that circulate in the blood and repair damaged endothelium. The number and function of EPCs is predictive of endothelial function and cardiovascular events. Herein we discuss the impact of individual risk factors on EPC numbers and discuss the potential utility of EPC number as a cardiovascular risk-assessment tool that integrates traditional and emerging cardiovascular risk factors.

#### **2. The systemic basis of cardiovascular disease**

Cardiovascular disease the leading cause of mortality in the Western world and manifests as coronary disease, peripheral vascular disease, or ischemic stroke depending on the vascular territory affected. The ageing population and projected increases in prevalence and costs of care have highlighted the need for more effective prevention of cardiovascular disease (Heidenreich, Trogdon et al. 2011). These manifestations of cardiovascular disease share common risk factors of age, hypertension, diabetes, hypercholesterolemia and smoking (Roger, Go et al. 2011). Endothelial dysfunction is the precursor lesion to atherosclerosis and reflects depressed nitric oxide (NO) release from the endothelium (Furchgott 1996; Valgimigli, Merli et al. 2003). Basal release of NO from the endothelium regulates vascular tone and antagonizes the actions of vasoconstrictor substances. Further, NO possesses antiplatelet actions and down-regulates adhesion molecules that attract inflammatory cells to the endothelium (Deanfield, Halcox et al. 2007). The degree of endothelial dysfunction shows a graded response to the number of cardiac risk factors present (Bonetti, Lerman et al. 2003; Davignon and Ganz 2004) and is predictive of clinical events (Bonetti, Lerman et al. 2003; Davignon and Ganz 2004; Deanfield, Halcox et al. 2007). Since endothelial dysfunction occurs systemically, the atherosclerotic process involves a large portion of the arterial tree before it becomes clinically manifest (Deanfield, Halcox et al. 2007). Patient presentations to a cardiologist, cardiac surgeon, vascular surgeon or stroke neurologist with clinically manifest atherosclerosis are typically preceded by decades of endothelial dysfunction and depressed vascular repair throughout the entire arterial bed (Ross 1993). The systemic

Endothelial Progenitor Cell Number: A Convergence of Cardiovascular Risk Factors 267

infarct size (Britten, Abolmaali et al. 2003). Reduced EPC migration to chemotaxins and reduced ability of human EPCs to effect neovascularization in animal hindlimbs has also been related to individual cardiovascular risk factors such as increasing age, hypertension, hypercholesterolemia, family history of CAD, smoking and high Framingham risk scores (Vasa, Fichtlscherer et al. 2001; Hill, Zalos et al. 2003; Heeschen, Lehmann et al. 2004; Schmidt-Lucke, Rossig et al. 2005; Wang, Gao et al. 2007). While EPCs can be harvested from bone marrow to treat myocardial ischemia (Britten, Abolmaali et al. 2003) or threatened limb ischemia (Comerota, Link et al. 2010) on an investigational basis, herein we focus on the impact of cardiovascular risk factors on EPCs and the potential utility of measuring EPC numbers for risk assessment in primary and secondary prevention. We discuss the impact of individual risk factors on EPC number with a focus on studies undertaken in human subjects and describe how risk factor control boosts EPC numbers. Each of the discussed risk factors individually suppresses EPC mobilization from the marrow and decreases peripheral survival making EPC number a universal risk factor (Hoenig, Bianchi et al. 2008).

Diabetes is a risk factor associated with heightened cardiovascular risk and endothelial dysfunction (De Vriese, Verbeuren et al. 2000; III 2002). In some series, diabetes has been associated with the same coronary risk as established coronary disease thereby making it a "coronary artery disease risk-equivalent" (Haffner, Lehto et al. 1998). Diabetics without manifest cardiovascular disease have decreased EPC numbers compared to age-matched controls (Tepper, Galiano et al. 2002) and diabetics with manifest macrovascular disease such as CAD, peripheral vascular disease or stroke have further reduced EPC numbers (Fadini, Miorin et al. 2005; Brunner, Hoellerl et al. 2011). Further, EPCs in diabetics are dysfunctional when compared to EPCs from non-diabetic subjects. The depressed EPC numbers in diabetes are thought to contribute to impaired collateralization of vascular ischemic beds (Waltenberger 2001) and may predispose this group to developing nonhealing diabetic ulcers which may be ameliorated by injecting EPCs into ischemic lower limb muscles (Huang, Li et al. 2005). Indeed, among diabetic patients with peripheral vascular disease, EPC numbers correlated negatively with the ankle brachial index and patients with ischemic ulcers had the lowest EPC numbers (Fadini, Miorin et al. 2005). Blood sugar levels are inversely correlated with EPC numbers implying a direct relationship between hyperglycemia and depressed EPC numbers (Fadini, Miorin et al. 2005). In the laboratory, hyperglycemia directly impairs EPC function by impairing the ability of these cells to migrate (Krankel, Adams et al. 2005). Diabetics with good glucose control have higher EPC numbers and more functional EPCs when compared to diabetics with poorly controlled glucose (Churdchomjan, Kheolamai et al. 2010) and treating newly-diagnosed diabetics with secretagogues increases EPC numbers and is associated with a concordant improvement in endothelial function (Kusuyama, Omura et al. 2006; Liao, Chen et al. 2010). Likewise, insulin-sensitizing agents such as pioglitazone or rosiglitazone boost EPC numbers and the increase in EPCs is correlated with the reduction in C-reactive protein and increase in adiponectin (Kusuyama, Omura et al. 2006; Makino, Okada et al. 2008). The inverse relationship between EPC numbers and HbA1c and insulin resistance indices implies that EPC numbers decline in pre-diabetic states such as the metabolic syndrome and insulin resistance (Tepper, Galiano et al. 2002; Penno, Pucci et al. 2011). Indeed, EPC

**4. Insulin resistance, the metabolic syndrome and diabetes**

nature of atherosclerosis is highlighted by the fact that >50% of patients with stroke or peripheral vascular disease have co-morbid atherosclerotic coronary disease (Hirsch, Haskal et al. 2006; Brott, Halperin et al. 2011) and patients with manifest disease in multiple arterial beds are at an increased risk of cardiovascular death and recurrent events (Steg, Bhatt et al. 2007). Since the description of circulating marrow cells that repair the endogenous arterial bed, "endothelial progenitor cells" (EPCs), an increasing research interest has been focused on how risk factors impact on the numbers of these cells and their ability to repair the vasculature and maintain endothelial function.

#### **3. Endothelial progenitor cells and atherosclerosis**

The modern concept that circulating marrow cells, EPCs, circulate in adult animals and repair the vasculature originates stems from the observation in the late 90s that marrowderived mononuclear cells circulate in adult animals and directly contribute to neovascularization in animal models of hindlimb ischemia, myocardial infarct remodeling and post-stroke neovascularization (Asahara, Murohara et al. 1997; Asahara, Masuda et al. 1999; Zhang, Zhang et al. 2002; Metharom and Caplice 2007). The clinical relevance of EPC numbers was brought to the forefront cardiovascular risk prognostication when EPC numbers were shown to correlate positively with flow mediated brachial artery reactivity (a measure of endothelial function) and inversely with the Framingham risk score (Hill, Zalos et al. 2003; Ghani, Shuaib et al. 2005; Chironi, Walch et al. 2007). Endothelial dysfunction observed in patients with cardiovascular disease or its risk factors may reflect a depressed ability to "renew" the endothelium from the circulating pool of EPCs which act to restore endothelial function. Indeed, patients with coronary artery disease (CAD) and stroke were shown to have EPC numbers that are reduced when compared to *age-matched* healthy volunteers (Vasa, Fichtlscherer et al. 2001; Lambiase, Edwards et al. 2004; Ghani, Shuaib et al. 2005). EPC numbers, which are usually assessed by flow cytometry for CD34+KDR+ cells, carry prognostic significance in patients with and without cardiovascular disease. EPC numbers predict clinical events in patients with established CAD. Amongst patients with CAD, lower EPC numbers were associated with increased severity of CAD and higher risks of death from cardiovascular causes, major cardiovascular events, revascularization or hospitalization (Schmidt-Lucke, Rossig et al. 2005; Werner, Kosiol et al. 2005; Kunz, Liang et al. 2006; Wang, Gao et al. 2007). In asymptomatic individuals, EPC numbers correlate with the number of vascular beds with subclinical disease. In a study using ultrasound to characterize disease in the carotid artery, abdominal aorta and femoral artery, the number of EPCs cells was shown to be decreased stepwise in patients with plaque in 0, 1, 2 and 3 of the sites (Chironi, Walch et al. 2007). Further, EPC numbers correlate with cardiovascular disease surrogates such as carotid intima-media thickness even after correction for the Framingham risk score and C-reactive protein (Fadini, Coracina et al. 2006).

In addition to absolute EPC numbers, the functional capacity of EPCs in repairing the vasculature is impaired by cardiac risk factors. EPCs harvested from the marrow of human patients with ischemic cardiomyopathy show an impaired capacity to effect neovascularization and incorporate into the vasculature in a mouse hindlimb ischemia model (Heeschen, Lehmann et al. 2004). In a human trial testing the efficacy of EPCs in repairing the coronary vasculature after a re-perfused myocardial infarction, the migratory capacity of EPCs to chemotaxins was the strongest multivariate predictor of reduction in

nature of atherosclerosis is highlighted by the fact that >50% of patients with stroke or peripheral vascular disease have co-morbid atherosclerotic coronary disease (Hirsch, Haskal et al. 2006; Brott, Halperin et al. 2011) and patients with manifest disease in multiple arterial beds are at an increased risk of cardiovascular death and recurrent events (Steg, Bhatt et al. 2007). Since the description of circulating marrow cells that repair the endogenous arterial bed, "endothelial progenitor cells" (EPCs), an increasing research interest has been focused on how risk factors impact on the numbers of these cells and their ability to repair the

The modern concept that circulating marrow cells, EPCs, circulate in adult animals and repair the vasculature originates stems from the observation in the late 90s that marrowderived mononuclear cells circulate in adult animals and directly contribute to neovascularization in animal models of hindlimb ischemia, myocardial infarct remodeling and post-stroke neovascularization (Asahara, Murohara et al. 1997; Asahara, Masuda et al. 1999; Zhang, Zhang et al. 2002; Metharom and Caplice 2007). The clinical relevance of EPC numbers was brought to the forefront cardiovascular risk prognostication when EPC numbers were shown to correlate positively with flow mediated brachial artery reactivity (a measure of endothelial function) and inversely with the Framingham risk score (Hill, Zalos et al. 2003; Ghani, Shuaib et al. 2005; Chironi, Walch et al. 2007). Endothelial dysfunction observed in patients with cardiovascular disease or its risk factors may reflect a depressed ability to "renew" the endothelium from the circulating pool of EPCs which act to restore endothelial function. Indeed, patients with coronary artery disease (CAD) and stroke were shown to have EPC numbers that are reduced when compared to *age-matched* healthy volunteers (Vasa, Fichtlscherer et al. 2001; Lambiase, Edwards et al. 2004; Ghani, Shuaib et al. 2005). EPC numbers, which are usually assessed by flow cytometry for CD34+KDR+ cells, carry prognostic significance in patients with and without cardiovascular disease. EPC numbers predict clinical events in patients with established CAD. Amongst patients with CAD, lower EPC numbers were associated with increased severity of CAD and higher risks of death from cardiovascular causes, major cardiovascular events, revascularization or hospitalization (Schmidt-Lucke, Rossig et al. 2005; Werner, Kosiol et al. 2005; Kunz, Liang et al. 2006; Wang, Gao et al. 2007). In asymptomatic individuals, EPC numbers correlate with the number of vascular beds with subclinical disease. In a study using ultrasound to characterize disease in the carotid artery, abdominal aorta and femoral artery, the number of EPCs cells was shown to be decreased stepwise in patients with plaque in 0, 1, 2 and 3 of the sites (Chironi, Walch et al. 2007). Further, EPC numbers correlate with cardiovascular disease surrogates such as carotid intima-media thickness even after correction for the

vasculature and maintain endothelial function.

**3. Endothelial progenitor cells and atherosclerosis**

Framingham risk score and C-reactive protein (Fadini, Coracina et al. 2006).

In addition to absolute EPC numbers, the functional capacity of EPCs in repairing the vasculature is impaired by cardiac risk factors. EPCs harvested from the marrow of human patients with ischemic cardiomyopathy show an impaired capacity to effect neovascularization and incorporate into the vasculature in a mouse hindlimb ischemia model (Heeschen, Lehmann et al. 2004). In a human trial testing the efficacy of EPCs in repairing the coronary vasculature after a re-perfused myocardial infarction, the migratory capacity of EPCs to chemotaxins was the strongest multivariate predictor of reduction in infarct size (Britten, Abolmaali et al. 2003). Reduced EPC migration to chemotaxins and reduced ability of human EPCs to effect neovascularization in animal hindlimbs has also been related to individual cardiovascular risk factors such as increasing age, hypertension, hypercholesterolemia, family history of CAD, smoking and high Framingham risk scores (Vasa, Fichtlscherer et al. 2001; Hill, Zalos et al. 2003; Heeschen, Lehmann et al. 2004; Schmidt-Lucke, Rossig et al. 2005; Wang, Gao et al. 2007). While EPCs can be harvested from bone marrow to treat myocardial ischemia (Britten, Abolmaali et al. 2003) or threatened limb ischemia (Comerota, Link et al. 2010) on an investigational basis, herein we focus on the impact of cardiovascular risk factors on EPCs and the potential utility of measuring EPC numbers for risk assessment in primary and secondary prevention. We discuss the impact of individual risk factors on EPC number with a focus on studies undertaken in human subjects and describe how risk factor control boosts EPC numbers. Each of the discussed risk factors individually suppresses EPC mobilization from the marrow and decreases peripheral survival making EPC number a universal risk factor (Hoenig, Bianchi et al. 2008).

#### **4. Insulin resistance, the metabolic syndrome and diabetes**

Diabetes is a risk factor associated with heightened cardiovascular risk and endothelial dysfunction (De Vriese, Verbeuren et al. 2000; III 2002). In some series, diabetes has been associated with the same coronary risk as established coronary disease thereby making it a "coronary artery disease risk-equivalent" (Haffner, Lehto et al. 1998). Diabetics without manifest cardiovascular disease have decreased EPC numbers compared to age-matched controls (Tepper, Galiano et al. 2002) and diabetics with manifest macrovascular disease such as CAD, peripheral vascular disease or stroke have further reduced EPC numbers (Fadini, Miorin et al. 2005; Brunner, Hoellerl et al. 2011). Further, EPCs in diabetics are dysfunctional when compared to EPCs from non-diabetic subjects. The depressed EPC numbers in diabetes are thought to contribute to impaired collateralization of vascular ischemic beds (Waltenberger 2001) and may predispose this group to developing nonhealing diabetic ulcers which may be ameliorated by injecting EPCs into ischemic lower limb muscles (Huang, Li et al. 2005). Indeed, among diabetic patients with peripheral vascular disease, EPC numbers correlated negatively with the ankle brachial index and patients with ischemic ulcers had the lowest EPC numbers (Fadini, Miorin et al. 2005). Blood sugar levels are inversely correlated with EPC numbers implying a direct relationship between hyperglycemia and depressed EPC numbers (Fadini, Miorin et al. 2005). In the laboratory, hyperglycemia directly impairs EPC function by impairing the ability of these cells to migrate (Krankel, Adams et al. 2005). Diabetics with good glucose control have higher EPC numbers and more functional EPCs when compared to diabetics with poorly controlled glucose (Churdchomjan, Kheolamai et al. 2010) and treating newly-diagnosed diabetics with secretagogues increases EPC numbers and is associated with a concordant improvement in endothelial function (Kusuyama, Omura et al. 2006; Liao, Chen et al. 2010). Likewise, insulin-sensitizing agents such as pioglitazone or rosiglitazone boost EPC numbers and the increase in EPCs is correlated with the reduction in C-reactive protein and increase in adiponectin (Kusuyama, Omura et al. 2006; Makino, Okada et al. 2008). The inverse relationship between EPC numbers and HbA1c and insulin resistance indices implies that EPC numbers decline in pre-diabetic states such as the metabolic syndrome and insulin resistance (Tepper, Galiano et al. 2002; Penno, Pucci et al. 2011). Indeed, EPC

Endothelial Progenitor Cell Number: A Convergence of Cardiovascular Risk Factors 269

(Umemura, Soga et al. 2008). Similarly, treatment of diabetics with angiogentinsin receptor blockers boosts EPC numbers (Bahlmann, de Groot et al. 2005). Treating patients with an angiotensin-converting enzyme (ACE) inhibitor such as ramipril has similar effects (Bahlmann, de Groot et al. 2005). Angiotensin II reduces the proliferative capacity of cultured EPCs and induces cell death (Imanishi, Hano et al. 2005). Such observations may explain why drugs such as ACE inhibitors may have beneficial effects that are greater than

Hypercholesterolemia is a pivotal cardiovascular risk factor and much there is much focus on treating this risk factor (ATP III 2002). Low density lipoprotein cholesterol (LDL-C) is the primary treatment target in both primary and secondary prevention of cardiovascular disease and there is a log-linear relationship between LDL-C level and CAD risk (ATP III 2002). LDL-C is inversely correlated with EPC number and function in human patients (Chen, Zhang et al. 2004). Statin therapy has been shown to increase EPC numbers and function (Fadini, Albiero et al. 2010; Jaumdally, Goon et al. 2010) and to enhance EPC numbers in response to ischemic stimuli (Spadaccio, Pollari et al. 2010; Hibbert, Ma et al. 2011). The improvement of endothelial function associated with statin use is directly correlated with the increase in EPC numbers and measures of EPC function (Higashi, Matsuoka et al. 2010). Similarly, lipid apheresis for resistant hypercholesterolemia improves EPC function and mobilization (Patschan, Patschan et al. 2009; Ramunni, Brescia et al. 2010). Low high density lipoprotein cholesterol (HDL-C) has been identified as secondary therapeutic target and reconstituted HDL-C infusion improves endothelial function and

Inflammatory conditions such as rheumatoid arthritis (RA) have, relative to traditional risk factors, been only recently associated with an increased cardiovascular risk. Like other cardiovascular risk factors, RA is associated with endothelial dysfunction (Herbrig, Haensel et al. 2006). Patients with RA have a life expectancy that is reduced by 5-10 years and the excess mortality is from cardiovascular disease which is increased roughly 4-fold (Wrigley, Lip et al. 2010). RA is particularly associated with a virulent form of coronary atherosclerosis characterized by high coronary artery calcium scores. However, the patients with RA that are at particular cardiovascular risk are those with active disease and high disease activity scores (Grisar, Aletaha et al. 2005). EPC numbers and EPC proliferative capacity show an inverse correlation with disease activity scores (Grisar, Aletaha et al. 2005; Herbrig, Haensel et al. 2006; Egan, Caporali et al. 2008). The increased risk of cardiovascular events is not limited to RA and has been described in other inflammatory states such as systemic lupus erythematosus (SLE) (Urowitz, Bookman et al. 1976; Roman, Shanker et al. 2003), human immunodeficiency virus (HIV) infection (van Leuven, Sankatsing et al. 2007), inflammatory bowel disease (Danese and Fiocchi 2003) or periodontitis (Mattila, Nieminen et al. 1989). Pre-menopausal women with SLE have a risk of myocardial infarction that is increased a staggering 50-fold compared to healthy controls (Manzi, Meilahn et al. 1997). SLE is associated with impaired EPC function and hence a decreased capacity to repair the endothelium (Deng, Li et al. 2010; Ablin, Boguslavski et al. 2011). Inflammatory conditions

the observed reduction in blood pressure (Yusuf, Sleight et al. 2000).

raises EPC numbers (Nieuwdorp, Vergeer et al. 2008).

**8. Inflammatory conditions**

**7. Dyslipidemia**

numbers decrease as more metabolic syndrome criteria are met (Fadini, de Kreutzenberg et al. 2006; Jialal, Devaraj et al. 2010) and are also decreased in other pre-diabetic states such as gestational diabetes (Penno, Pucci et al. 2011) or the polycystic ovarian syndrome (Dessapt-Baradez, Reza et al. 2011). Given that EPC numbers repair the vasculature and maintain endothelial dysfunction, this decreased capacity for repair of the vasculature may provide a mechanism for the increased risk of cardiovascular events observed in patients with the metabolic syndrome (Mottillo, Filion et al. 2010).

#### **5. Gender and age**

Age and male gender are irreversible cardiovascular risk factors. Healthy middle-aged women have higher EPC numbers than men (Hoetzer, MacEneaney et al. 2007). Young men have similar EPC numbers as post-menopausal women and this may explain why men are prone to cardiovascular disease at a younger age. Women, on average tend to develop cardiovascular disease after menopause with an incidence that equals that of age-matched men 10 years after the menopause. This time in a woman's life, 10 years after the menopause, is associated with a decrease in EPC numbers and EPC function (Bulut, Albrecht et al. 2007; Rousseau, Ayoubi et al. 2010). This decline in EPC numbers may be due to the lack of estrogen since hyperestrogenic states (e.g. during ovarian stimulation) have been shown to be associated with an increase in EPC numbers and there is a normal variation with the ovarian cycle (Rousseau, Ayoubi et al. 2010). Hormone replacement therapy can boost EPC numbers in postmenopausal females by 25% (Bulut, Albrecht et al. 2007) and enhance endothelial function (Sanada, Higashi et al. 2003; Kalantaridou, Naka et al. 2006).

Ageing is associated with endothelial dysfunction and dysfunctional EPCs that are more prone to apoptosis and have reduced proliferative capacity (Heiss, Keymel et al. 2005; Kushner, Maceneaney et al. 2011). Further, the elderly are less able to mobilize EPCs in response to ischemic stimuli (Scheubel, Zorn et al. 2003). With ageing, the endothelial progenitor cells have shortened telomeres, which are the repetitive DNA at the ends of chromosomes that protect DNA integrity (Kushner, Van Guilder et al. 2009). Telomere shortening has been described in patients with CAD compared to healthy controls (Ogami, Ikura et al. 2004). Hence, this may provide a mechanism whereby EPCs from elderly individuals are more likely to undergo proliferative senescence and an increased susceptibility to apoptosis which can contribute to decreased EPC numbers. This generally occurs around the age of 55 which is temporally associated with the increased period of cardiovascular risk within a human's lifetime (Kushner, Van Guilder et al. 2009). Hence, the ability to generate functional EPCs, to rejuvenate the endothelium lining the arteries and maintain endothelial function may be key in the pathogenesis of cardiovascular disease with aging.

#### **6. Hypertension**