Dietary Nitrate Supplmentation in Peripheral Artery Disease and Diabetes Mellitus+Peripheral Artery Disease

Peripheral artery disease (PAD) is a form of cardiovascular disease (CVD) caused by atherosclerotic occlusions in the legs. It affects approximately 5% of the US population over 50 years of age, one third of which suffer from ischmic leg pain that occurs with walking and improves with rest. We have previously shown that supervised exercise therapy is an efficacious treatment for PAD, with improvements of 66% in claudication pain onset time and 17% in peak walking time after 3 months of supervised training.  Despite these improvements, claudication pain is still the major limiting factor to the intensity and duration of work that can be performed during training for daily activities. 

Diminished nitric oxide (NO) bioavailibility is an underlying feature of CVD and PAD. NO plays an important role in inflammation, thrombosis, vascular tone, blood flow, tissue perfusion, and exercise capacity. Consequently, a therapy that would effectively deliver NO to the peripheral tissues that are under-perfused due to occlusive vascular disease is an area of our interested.  

Our aim is to utilize the dietary supplmentation of nitric oxide (in the form of beetroot juice) as an intervention to acutely improve oxygenation to areas of ischema AND to chronically increase vessel growth to these ischemic areas. We are currently working with two populations to investigate the potential benefits of this intervention and treatment method.

• Increased Plasma Nitrite, Tissue Oxygenation and Functional Changes in PAD (1R21 1HL 111972-01)
• Dietary Nitrate to Augment Exercise Training Benefits in DM+PAD (1 R21 HL113717-01)

Incidence of PAD in subjects with Type II Diabetes is greatly increased in comparison to non-diabetic individuals. It is thought that diabetics may be less able to up-regulate endothelial NO production during acute exercise and in response to chronic training.  Therefore, increasing dietary nitrite supplementation may hold significant potential as an effective, selective NO donor for acute and chronic tissue ischemia and a means of effective therapy for individuals with both diabetes and PAD.

Through these interventions, we hope to improve exercise tolerance, overall function, and quality of life for these individuals with PAD as well as contribute valuable treatment possibilities to the medical community.

Acoustic Radiation Force Imaging of Carotid Plaque Morphology and Composition NHLBI-2RO1HL075485

In conjunction with Duke biomedical engineers Gregg Trahey (PI) and Jeremy Dahl, and vascular surgeons Jeffrey Lawson and Ban Sileshi, we are developing and testing acoustic radiation force impulse (ARFI) imaging for detection of vulnerable carotid artery plaques.

ARFI imaging is a new ultrasonic imaging method that uses short duration (.03 - 1 meters per second) acoustic radiation force to generate localized displacements in tissue. These displacements are tracked using conventional ultrasound and correlation-based speckle-tracking methods. Tissue displacement is inversely related to tissue stiffness, and the recovery of tissue to its original position is related to its viscoelastic properties.

Because arterial walls, soft tissue, atheromas, and calcifications have a wide range in stiffness, they represent excellent candidates for ARFI imaging. Furthermore, because a single transducer on a diagnostic scanner is used to both generate the radiation force and track the resulting displacements, and there are thousands of installed ultrasonic scanners in vascular laboratories that could be programmed to include ARFI, this modality could quickly and inexpensively be added to current vascular diagnostic methods. The aim of this grant is to further develop the tecnology of ARFI and to evaluate the potential of ARFI imaging for the generation of high-resolution images of carotid plaque composition and detecting vulnerable plaques.

Above is a longitudinal view of a stiff, possibly fibrous, plaque in the common carotid artery of a 49-year-old male. The B-mode image indicates a plaque on the far wall of the common carotid artery. The plaque appears to wrap around a significant portion of the artery (for cross-section scans, not shown here), and is therefore visible on the near wall of the common carotid artery. The plaque appears to be homogeneously stiff throughout.

In contrast, below is an example of an apparently heterogeneous plaque containing a soft region surrounded by a stiff cap in the carotid bifurcation of a 55-year-old female volunteer. The plaque is located near the bifurcation of the carotid artery and is visible on both the proximal and distal walls. The soft region of the plaque is visible on the distal wall as the oval shaped region extending from 23 to 25 mm depth and -2 to 4 mm laterally in the ARFI image.

The soft region is surrounded by a significantly stiffer region. The displacement of the soft region ranges from 2.5 - 4 µm, compared to 0.5 - 1 µm in the surrounding stiffer tissue. The surrounding tissue is approximately 1.3 mm thick between the soft region and the vessel lumen, except on the left side, where the thickness of the cap decreases to approximately 0.7 mm. At 0.7 mm, the stiff cap may be considered to be at the upper limit of the range indicating vulnerability (Ge et al., 1999). Ge et al. (1999) also noted that a lipid core with area greater than 1 mm2or a core-to-plaque ratio greater than 20 percent also correlated well with rupture. The soft region in this plaque meets Ge’s definition of a vulnerable plaque, assuming that this region is a lipid core.

We are also working toward development of 3-D modeling of carotid arteries and plaques to enable us to match accurately with histology samples.

Click here for further information about our previous research studies.

The biochemistry laboratory identifies metabolites of nitric oxide bioavailability and reactive oxygen species applied to endothelial and vascular health.

Nitric oxide (NO) is a simple diatomic molecule and a diffusible gas. It is unstable and has a half-life of seconds. It has been shown to play a role in the regulation of almost every major organ system.

In the vasculature, NO is synthesized by the endothelium and can also be carried by red blood cells. A simplified map of possible NO pathways in the vasculature is shown below.

NO has several anti-atherogenic properties, including:

• Inhibition of platelet aggregation (anti-thrombotic)
• Inhibition of leucocyte adhesion to the vessel wall (anti-inflammatory)
• Inhibition of smooth muscle growth into the lumen (anti-proliferative)
• Inhibition of vasoconstrictors (vasodilation-indirect)
• Vasodilation (via shear and/or receptor mediators) 

NO in the Plasma

We have investigated the effects of vascular health on NO metabolites. Plasma nitrite (NO2-) is the main oxidation product of NO and has been shown to reflect changes in eNOS activity. It therefore appears to be a good marker of recent NO bioavailability.

Example of a Recent Study

Abbreviations:

• RF = greater than two traditional risk factors, no clinically diagnosed CVD
• DM = type 2 diabetes no clinically diagnosed CVD
• PAD = intermittent claudication > three months and ABI<0.9 at rest. 
• (In Press, Nitric Oxide: Biology and Chemistry, 2009)

We have shown plasma NO2- can be upregulated following occlusion (five minutes) and reactive hyperemia in the arm. In 15 apparently healthy subjects (34.1+7.3yrs) mean plasma nitrite concentrations were 415+64.0nM at baseline, and 634+57.1nM following reactive hyperemia. This represents a 52.5 percent increase in plasma nitrite (p=0.015). Perhaps, just as significant as the increase in the mean plasma nitrite concentration is the observation that of the 15 individuals who performed the test, the plasma nitrite level fell in only two (and in one of those by less than 5 percent). There was no change in plasma NOx (predominantly NO3-) levels.

Additionally, we have measured plasma NO2- in several different populations with varying severity of vascular disease or risk. We use acute exercise stress as a stimulus to test if individuals can upregulate NO.

There were no differences in resting plasma nitrite values between groups. However, following the GXT, the RF group had significantly greater plasma nitrite concentrations than the other groups (after adjustment for both age and VO2peak). Furthermore, within group (pre-post) t-tests revealed a significant increase in plasma nitrite in the RF group (+39.3 percent), no change in the DM (-15.51 percent), and a significant decrease in the PAD (-44.20 percent) and PAD+DM (-39.95 percent). There were no significant changes in plasma nitrate from baseline for any of the groups following treadmill exercise. 

Exercise training has been shown to improve endothelial function, so we are currently examining its effects on nitrite response to an acute exercise stress (nitrite flux) in subjects with various degrees of vascular disease. In particular we are looking at PAD and diabetic subjects. The improvement in "nitrite flux" is shown in the figure below. This was related to changes in physiological endothelial function and exercise performance.

(This work is supported in part by NIHLBI grant 5RO1 HL-075752-05.)

NO in the Red Blood Cell

James group in Cardiff (Wales) have previously shown there may be defects in NO offloading in glycated RBC's (found in Diabetes). We have similar findings, and the pilot data opposite suggests that glycation (HbA1c11.3 percent) moves the free O2 binding curve to the left in comparison to normal Hb (A1c 5.4 percent), indicating a change from R to T state at lower pO2 and “later” O2 offloading.

We also measured the physiological responses of this glycation effect. Below is data from a single rabbit aortic ring suspension trial (three rings per condition). In the presence of glutathione (GSH), normally glycated Hb (A1c5.4 percent) dilated hypoxic vessel rings both prior to (9 percent) and following SNO loading (18.5 percent). Under identical conditions there was no response from highly glycated Hb (A1c11.8 percent). Taken together, these findings suggest that in glycated cells, NO may be "stuck" on the cell, which may effect delivery at the micro-vessels.

The loss of this “RBC-NO carrier” system may be very pertinent for vascular disease prevention in diabetic subjects.

This ongoing work is funded by the 7-08-IN-01 American Diabetes Association Innovative Award.

Staff

Thomas Stabler is responsible for the day-to-day running of the lab.

Contact Information

Office: Building GSRB1, Room 1033, 595 LaSalle Street, Durham, NC, 27710
Phone: 919-681-6808
E-mail: thomas.stabler@duke.edu

The vascular lab uses non-invasive techniques to assess markers of endothelial and vascular health.

We routinely perform assessments of both physiological and structural markers of vascular health for our own research projects and as a service for other investigators at Duke and externally. Some of these procedures as well as data from our research are detailed below.

Some of the procedures we employ include:

Physiological Markers

Physiological markers of vascular disease are dynamic measures that can be influenced by stress or medications. Consequently they are usually measured in the mornings following an overnight fast and other preparations.

• Brachial artery flow-mediated dilation
• Pulse wave velocity (PWV) and pulse wave reflection (PWR) analysis of vascular stiffness
• Plethysmographic limb blood flow

Structural Markers

• Carotid intimal-medial thickness (CIMT)
• Doppler ankle-brachial index (ABI)

Physiological Markers

Brachial Artery Flow-Mediated Dilation

Brachial artery flow-mediated dilation (BAFMD) is a dynamic assessment of endothelial function, measured in response to forearm reactive hyperemia. It has been associated with nitric oxide bioavailability and shown to be prognostic in coronary artery disease and peripheral arterial disease. The response of the brachial artery has been shown to be similar to that of the coronary arteries but is much easier to measure and so is often used as a surrogate measure.

Above is a typical image of the brachial artery with specialized software tracking the vessel wall over time. We have also set image acquisition to only capture diastole images.

The figures below (left) show the diameter responses over time of healthy subjects versus subjects with CVD to the hyperemic stimulus. Notice the healthy subjects dilate over 5 percent, whereas the CVD group peak at 2 percent. This is reflected (right) in the bar graph of responses from different disease states.
(H = healthy, RF = CVD risk factor subjects, DM = type 2 diabetics, PAD = peripheral arterial disease)

Cardiovascular disease (CVD) risk factors associated with abnormal BAFMD (and reduced NO bioavailability) include:

• Hypercholesterolemia/hyperlipidemia
• Hypertension
• Diabetes/hyperglycemia
• Aging
• Cigarette smoking
• Oxidative stress
• Sedentary lifestyle

Pulse wave velocity (PWV) and reflection (PWR) analysis of vascular stiffness

Pulse Wave Reflection

The blood pressure waveform is the sum of the pressure wave generated by the left ventricle and the pressure waves reflected from terminations and bifurcations in vascular beds.

Stiffer arteries (the aorta and muscular arteries) increase the speed of the traveling pressure waves, leading to earlier return to the heart of the reflected waves.

Vasodilation and vasoconstriction of the muscular arteries changes both the size and speed of the reflected waves.

An earlier return of the reflected pressure wave changes the pressure waveform by increasing the central systolic and pulse pressures. In the aorta, the reflected wave moves also more into systole.

These changes may have important clinical implications, including:

Central pulse pressure increases, increasing risk of stroke and renal failure (increasing arrows)
Left ventricle (LV) load increases, increasing LV mass, and accelerating progress towards LV hypertrophy and heart failure (increasing arrows)
Coronary artery perfusion pressure in diastole decreases, increasing risk of myocardial ischemia (decreasing arrows)

Pulse Wave Velocity

Pulse wave velocity is a direct measure of arterial stiffness and has been shown in large populations to predict coronary artery disease and stroke. Pulse waves are waves that push against the artery walls with every heartbeat as blood moves through arteries to supply body tissues. Pulse wave velocity is the speed with which one wave or pulse travels from the heart through the arteries of the body.

Typically, the faster the pulse wave travels, the stiffer the arteries, and vice-versa. For example, a pulse wave velocity of around six metres per second (m/s) is considered highly compliant (a healthy arterial system), whereas velocities in excess of 14 m/s indicate stiffer arteries (a less healthy arterial system).

​Plethysmographic Limb Blood Flow (Arterial and Venous Function)

We use strain gauge plethysmography to look at whole limb arterial inflow, venous capacitance, and maximum venous outflow. This test detects possible blockages in arteries, endothelial dysfunction, and deep venous thrombosis in the legs.

Structural Markers of Vascular Disease

Carotid intimal-medial thickness

Carotid intimal-medial thickness (CIMT) levels have been shown to predict cardiovascular events such as heart attack and stroke.

We take multiple measures of the IMT in multiple sections of the carotid arteries at several different angles of interrogation. Using specialized software to measure the artery lining and data from the Atherosclerosis Risk in Communities trial (ARIC), we can estimate the rank score of individuals’ CIMT compared with data from people with of the same age, race and gender.

CIMT Prediction Trials

Atherosclerosis Risk in Communities (ARIC) study

• 7289 females, 5552 males, 45-70 years, no history of coronary artery disese, followed four to seven years
• Hazard ratio for myocardial infarction of coronary heart disease high versus low tertiles were 6.69 for females and 2.88 for males

Cardiovascular Health Study

• 5858 subjects, older than 65 years, no apparent coronary heart disease, followed for 6.2 years
• Highest quintile = 3.87RR of myocardial infarction or stroke versus lowest
• Strong predictor even after adjustment for risk factors

Doppler Ankle-Brachial Index (ABI)

This test is used to diagnose peripheral artery disease. It compares blood pressures in the feet with those in the arms to find out if there is a reduction in perfusion in the lower limbs. A normal ABI is 1.0 or greater (with a range of 0.90 to 1.30).

Staff

Katherine L Ham, MS 
Phone: 919-660-6793
E-mail: katherine.ham@duke.edu 

Mitch VanBruggen, MA
Phone: 919-660-6638
E-mail: mitch.vanbruggen@duke.edu 

Contact Information

Office: The Andrew Wallace Clinic Building
3475 Erwin Road, Durham, NC, 27710