Passive Limb Movement: A Tool to Assess Vascular Health and Guide Rehabilitation

BACKGROUND AND SIGNIFICANCE:

Importance and impact: It is becoming increasingly apparent that arterial endothelial
dysfunction contributes significantly to the age-related downward spiral that includes
mobility limitation, frailty, and ultimately cardiovascular disease (CVD) endothelial
dysfunction has been documented to manifest in the coronary epicardial and resistance
vasculature as well as the peripheral arteries, indicating that such dysfunction is a
systemic condition. Indeed, there is a growing appreciation that the vast majority of CVD is,
in fact, associated with endothelial dysfunction. Thus, developing a better understanding of
the relationship between arterial aging and CVD is one of the most important clinical
challenges that the investigators currently face. The vascular endothelium, a single layer of
cells lining the blood vessel wall, plays an essential role in maintaining both arterial
function and health. In either an autocrine or paracrine fashion, vascular endothelial cells
synthesize and release numerous biologically active molecules that modulate arterial
structure as well as vasodilatory, thrombolytic, and vasoprotective functions. The transition
from normal endothelial function to the phenotype of endothelial dysfunction contributes to
the development of atherosclerosis and increased CVD risk. Specifically, these vascular
changes catalyzed by diminished nitric oxide (NO) bioavailability lead to vasoconstriction
and an endothelial state that is pro-coagulative, pro-proliferative, and pro-inflammatory.
The recognition that the endothelium is not simply a passive interface between blood and
vessel wall, but is also the site of NO production, has led to great advances in the field of
vascular research. Indeed, endothelial derived NO is now not only recognized as one of the
most important vasodilators, particularly in the muscular arteries, but it is apparent that
NO also inhibits other key events associated with the development of atherosclerosis such as
leukocyte adhesion and migration, platelet adhesion and aggregation, and smooth muscle cell
proliferation. Thus, a reduction in NO bioavailability is evidence of a broadly dysfunctional
endothelial phenotype, and therefore the assessment of NO-mediated vasodilatory capacity may
provide significant insight into the integrity and function of the endothelium. Although the
process of CVD can begin early in life and is accelerated by the aging process, endothelial
dysfunction, a recognized precursor to atherosclerosis, precedes the structural changes
associated with this disease and so can be used as a predictor of CVD onset.

Originality:

Most, if not all, CVD risk factors are associated with endothelial dysfunction and risk
factor modification can lead to improved vascular function. However, although, over the past
20 years, several methodologies have been developed to assess endothelial function in humans,
and these approaches have contributed extensively to clinical research, none have been
adopted into clinical practice. In fact, not a single method for assessing endothelial
function has been recommended by clinical guidelines for the planning of primary or secondary
prevention of CVD. The novel passive leg movement (PLM)-induced increase in blood flow
approach to assess endothelial function may be the clinically relevant tool that has long
been missing from the healthcare provider's arsenal.

Contribution to Veterans Health Administration (VHA): VA demographics reveal a population
that is significantly skewed toward the older sector and thus heavily burdened by
age-associated CVD. Even in the population at large it is estimated that by the year 2050
over 21% of people will be over 60 years of age. Although these data are impressive, the
Veteran population is far ahead of these predictions with 51% (12.3 million) of Veterans
already over 60 years of age. As advancing age is the major risk factor for CVD, the risk of
CVD increases progressively across the lifespan and although death rates as a result of CVD
have fallen over the last 50 years, CVD remains the leading cause of morbidity and mortality
in the U.S. Given the already accelerated aged demographics in the VA Health Care System and
the strong link between CVD and age, there is little doubt that the burden of CVD-related
healthcare costs within the VA will continue to grow. Thus, it is not surprising that aging
and mobility limitation, both of which are linked to endothelial dysfunction, are two
priority areas emphasized by the VA RR&D Service and are Medical Research Priorities for the
VHA system as a whole.

Post cuff-occlusion flow mediated vasodilation (FMD) as a test of NO bioavailability:

Prior to development of the novel PLM approach to assess endothelial function, perhaps the
most likely method to be adopted for routine clinical use was the post cuff-occlusion FMD
test, typically performed in the brachial artery. In 1992, Celermajer et al. introduced this
method of measuring the changes in conduit artery diameter following a period of circulatory
occlusion as a non-invasive approach for in vivo determination of endothelium-dependent
vasodilation in humans. In the more than two decades that have followed, the application of
FMD testing in clinically-based research has become widespread, a progression that is
exemplified by the adoption of this methodology into large-scale clinical trials, including
recent phases of the Framingham Heart Study. The FMD test is particularly appealing due, at
least in part, to a previous report revealing a correlation between vasodilatory capacity of
the peripheral and coronary arteries, implicating FMD as a potential surrogate measure of
coronary endothelial health. Further, it has been clearly demonstrated that FMD provides
independent predictive information for future cardiovascular events beyond traditional risk
factors. FMD testing has thus emerged as a straight-forward, non-invasive approach for
determining vascular function in health and disease. However, predominantly due to several
methodological deficiencies, this technique has never been embraced as a clinical tool.

Perhaps one of the most attractive aspects of FMD testing is that it has been purported to
provide an assay of vascular endothelial NO bioavailability. As the presence of this
endothelially-derived vasodilator has become synonymous with vascular health, the prospect of
quantifying NO in a non-invasive manner makes FMD testing particularly appealing for both
risk assessment and patient prognosis. The ability of NO synthase (NOS) inhibition with
L-NMMA to completely abolish radial and brachial artery FMD provided initial evidence that
FMD is governed primarily through the release of NO. However, in the face of significant
technical and methodological refinements in the field, recent studies have begun to challenge
the existing dogma regarding the NO-dependent nature of the FMD response. Indeed, the two
most recent studies in the radial artery have identified only minimal (0-33%) reduction in
FMD after the administration of L-NMMA, a NOS blocker, leaving considerable uncertainty as to
whether this test can still be relied upon to provide an index of NO bioavailability. Using
up-to-date measurement techniques and the typical site of assessment (brachial artery), the
investigators concluded that the majority of vasodilation provoked by brachial artery FMD
testing is not attributable to NO. In fact, the intra-arterial delivery of L-NMMA resulted in
only a modest (33%) reduction in FMD. Notably, L-NMMA administration also reduced the
post-occlusion hyperemic response, thereby diminishing the shear stimulus for brachial artery
vasodilation. When this effect was taken into account, FMD normalized for shear rate was not
different between control and L-NMMA conditions. These findings add to the growing
uncertainty surrounding the interpretation of FMD test results, the usefulness of this
approach clinically, and emphasize the need to develop a new and clinically relevant tool to
assess endothelial function.

Passive limb movement (PLM) as a test of nitric oxide (NO) bioavailability:

Originally, the group and others focused on the peripheral and central hemodynamic responses
to PLM movement as a reductionist model to better understand the factors controlling
movement-induced hyperemia. By removing the increase in metabolism, associated with active
exercise, important findings related to the control of skeletal muscle blood flow were
revealed. Specifically, in healthy humans, following the initial onset of passive movement,
there is a transient, yet robust, increase in leg blood flow and vascular conductance.
Through various experimental approaches the investigators have characterized the roles of
afferent feedback, the muscle pump, perfusion pressure, cardiac innervation/denervation, and
age in the PLM response. However, most germane to this proposal, in young healthy subjects,
the investigators have recently determined the role NO plays in the PLM-induced blood flow
response.

At rest NOS inhibition has consistently been documented to decrease limb blood flow and
vascular conductance by 30 to 40%, indicating an essential role of NO in controlling basal
blood flow. During active exercise the reduction in blood flow following NOS inhibition is
typically less than at rest, falling in the range of 5 to 25%. This implies a reduced
contribution of NO to exercise-induced hyperemia, an experimental paradigm characterized by
increased metabolism which also likely plays a significant role in elevating blood flow
during exercise. In contrast, the PLM-induced hyperemia, which does not invoke a peripheral
metabolic response, is intuitively more likely to be predominantly NO mediated. Indeed,
inhibition of NOS, with an intra-arterial L-NMMA infusion in the passively moved leg,
revealed that NO contributes to 80% of the hyperemia and vasodilation associated with PLM.
This robust and easily measured reduction in NO-mediated vasodilation and subsequent
hyperemia suggests that PLM-induced blood flow has significant promise as a new approach to
assess NO-mediated vascular function, an important predictor of CVD risk.

PLM and FMD directly compared:

Being noninvasive and with evidence of mirroring coronary endothelial function to some
degree, FMD is by far the most common approach to assess vascular function in clinical
research and has been in existence for over 20 years. However, FMD testing can be a somewhat
capricious endeavor exhibiting significant operator dependency, difficulty with performance,
a reliance upon very small vessel diameter changes, uncertainty about how to account for
varying shear stimuli, coupled with concerns regarding reproducibility, all combine to
explain why FMD has yet to be adopted clinically. In a recent article, highly germane to this
proposal, published in the Contemporary Reviews in Cardiovascular Medicine section of the
journal Circulation, Flammer et al. discuss many of these issues in their article entitled
"The assessment of endothelial function: from research to clinical practice". Interestingly,
they conclude that a clinical tool to assess endothelial function, suitable for daily
practice has yet to be established. Of note, PLM was not mentioned in this review as it is
truly in its infancy; however, PLM and FMD, the current front runner of available
methodologies, were compared for the criteria that were suggested to be essential for a
method that can assess endothelial function and be clinically relevant. As Flammer et al. did
not identify NO dependence in this list of criteria, but NO bioavailability is clearly a
major component of endothelial function, "NO dependence across the lifespan" was added to the
comparison. In addition to NO dependence across the lifespan, preliminary data showed PLM to
be superior to FMD in that it is reversible with interventions, mirrors coronary endothelial
function, improves risk stratification, and is reproducible. Other criteria in which PLM was
shown to be superior to FMD were that it is operator independent, and is easy to use.

From this side by side comparison of preliminary PLM data and FMD, it is apparent that if the
hypotheses in this proposal are proven to be correct, by rectifying most of the deficiencies
of FMD, endothelial function assessed by PLM has the makings of an easily adopted clinically
relevant tool.

PLM assessed endothelial function, CVD risk, and physical activity:

There is significant evidence that endothelial dysfunction is associated with CVD risk; in
fact, the more risk factors that are present, the worse are both the epicardial and
peripheral endothelial function. Endothelial dysfunction may, therefore, represent the effect
of these risk factors on vascular health. However, it is again important to recognize that
impaired endothelial function precedes structural changes and may therefore be considered "a
barometer" of the total cardiovascular risk burden of an individual. Thus, even a new method
with limited long-term data, which professes to allow the assessment of endothelial function,
must demonstrate some relation to CVD risk and risk factor modifications, such as increased
physical activity, that can alter CVD risk. Findings in the lab, although somewhat
preliminary, are evidence that endothelial function assessed by the blood flow response to
PLM, is, indeed, attenuated with increasing CVD risk and altered by physical activity.
Specifically, although there is significant variability within each group of subjects, which
would be expected and makes individual assessment so important, it is clear that the young
and old active subjects exhibit superior vascular function compared to their age-matched
sedentary counterparts as assessed by PLM. Additionally, although greater physical activity
can restore endothelial function in the old to that of the young, the elderly do not achieve
the same endothelial function of the young physically active subjects. Finally, the old
inactive subjects have the lowest endothelial function due to being male, as were all
subjects in this data set, being older (>70 yrs), and inactive. In terms of endothelial
function, as assessed by PLM, this group is closest to the patients with heart failure,
illustrating the ultimate consequence of numerous CVD risk factors and greatly attenuated
endothelial function. This clear link between the PLM-induced increased in blood flow and CVD
risk supports the concept that the development and ultimately the adoption of PLM as a tool
to non-invasively assess endothelial function could have a significant impact on both the VA
Health Care System and health care in general.

PRELIMINARY STUDIES:

Two major categories of specific aim are proposed, and preliminary data supporting the
approach and feasibility of each are presented below.

1. IS PASSIVE LIMB MOVEMENT (PLM) A RELIABLE AND NO-DEPENDENT ASSESSMENT OF VASCULAR
FUNCTION ACROSS THE HUMAN LIFESPAN?

The recognition that CVD and more specifically atherosclerosis is not simply a
structural disease, but has functional manifestations such as exaggerated
vasoconstriction due to a poorly functioning endothelium, has brought the need to assess
NO bioavailability to the forefront of vascular function testing in humans. Although FMD
was originally described as an in vivo bioassay of NO bioactivity, more recent studies
have questioned this capability. In contrast, current evidence suggests that PLM-induced
increases in blood flow are highly NO dependent. Specifically, the inhibition of NOS by
L-NMMA reduced the hyperemic response to continuous PLM, as assessed by area under the
curve (AUC), by 80%.

PLM, aging and NO: It is well recognized that vascular function and therefore NO
bioavailability is reduced with age and therefore if PLM is, indeed, a good indicator of
endothelial function it would be anticipated that the role of NO would be diminished in
the elderly population. In a group of 5 old subjects (>70 yr), compared to their young
counterparts, there was both a greatly attenuated PLM-induced hyperemia in the control
condition (delta peak 400 vs 700 ml/min) and a non-significant effect of NOS blockade.
These preliminary findings imply that endothelial function is diminished with age and
this can be explained by a reduction in NO bioavailability, both of which can be
documented by the use of PLM.

Single and continuous PLM:

Although, in young healthy subjects, the investigators have documented the highly NO
dependent nature of continuous PLM, single PLM remains largely unstudied. However,
single PLM is easier to perform, which makes it attractive clinically, and such an
approach, if eliciting a significant and reproducible hyperemic response, should limit
the potentially confounding central hemodynamic responses to PLM. Preliminary data,
collected in young healthy subjects (n=4), documents a robust PLM-induced increase in
blood flow, a slightly attenuated fall in mean arterial pressure, and avoidance of the
significant increase in cardiac output induced by continuous PLM, which some may
consider a confounding hemodynamic factor. Based upon these preliminary data, the PLM
approach to assess endothelial function looks very promising.

Reproducibility of PLM:

Any clinically relevant assessment tool needs to demonstrate reliability and therefore a
component of Specific Aim (1B) will be to document the reproducibility of PLM-induced
hyperemia. To achieve this goal, within subject comparisons of repeated single and
continuous PLM-induced hyperemic responses both within and between days will be
performed. Preliminary data illustrating this approach and, in this case, the net blood
flow (BF) assessed in response to continuous and single PLM performed on 3 separate days
on the same subject illustrate that the PLM-induced assessments typically exhibit good
reproducibility from day to day and perhaps suggest that single PLM may exhibit superior
reproducibility.

2. DOES PLM-ASSESSED PERIPHERAL VASCULAR FUNCTION REFLECT CORONARY VESSEL FUNCTION?

As vascular dysfunction is considered to be a systemic disease, it is expected that
endothelial function assessed in the periphery should "mirror" coronary endothelial
dysfunction. Therefore, the investigators will assess the correlations between the blood flow
response to single and continuous PLM, post occlusion FMD in the brachial and popliteal
arteries, and acetylcholine (ACh)-induced vasodilation in the left anterior descending (LAD)
coronary artery in the same subjects. This will allow an examination of the link between
peripheral vascular function and coronary vessel function, with a specific focus on the
PLM-induced hyperemia.

PRELIMINARY EVIDENCE OF A LINK BETWEEN PLM AND EXERCISE-BASED REHABILITATION The preliminary
data highlight the apparent impact of physical activity/inactivity (a CVD risk factor) on
endothelial function assessed by the PLM method. Specifically, both young and old physically
active subjects appear to exhibit a superior endothelial function than their inactive
counterparts. This bodes well for exercise as an approach to improve endothelial function,
but, more importantly, supports the use of the PLM-induced blood flow response as a sensitive
index of vascular function.

Maximal oxygen consumption (VO2max) assessed during a graded exercise test is currently one
of the strongest predictors of all-cause and CVD mortality and a very useful tool to assess
the efficacy of exercise-based rehabilitation. However, such maximal exercise testing is not
simple, requiring specific open circuit calorimetry equipment, physician coverage, and is not
without significant risk of an adverse event in patients with known CVD and the elderly
population. Interestingly and perhaps not completely surprisingly, recognizing the strong
link between endothelial function and CVD, preliminary data, collected thus far exclusively
in young healthy subjects, reveal that there is a relatively strong and statistically
significant relationship between VO2max and PLM-induced hyperemia. This observation suggests
that the assessment of the PLM-induced blood flow also holds significant promise as predictor
of all-cause mortality. However, unlike a maximal graded exercise test, PLM is without risk
and is completely independent of effort, which often confounds VO2max testing. In
combination, these preliminary data set the stage for the exciting evaluation of the, as of
yet, unexplored link between endothelial function assessed by PLM and exercise-based
rehabilitation.

To summarize, the preliminary data presented here clearly support the concept that PLM is a
reliable and NO-dependent assessment of vascular function across the lifespan, appears to
reflect coronary vessel health, and therefore would be a useful diagnostic tool in
rehabilitative medicine.

Inclusion Criteria:

- Young healthy subjects: No evidence of cardiovascular disease.

- Patients undergoing angiography: Clinical referral for an angiography.

Exclusion Criteria:

- Young healthy subjects: Evidence of cardiovascular disease.

- Patients undergoing angiography: Poor kidney function.