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

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 we 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 important role that nitric oxide (NO) plays in vasodilation. 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 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 also is apparent that NO 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, attenuated NO bioavailability is evidence of a broadly dysfunctional endothelial phenotype, and, therefore, the assessment of NO-mediated vasodilatory capacity provides 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 endothelial function. With the growing interest in personalized medicine, the development of diagnostic tools, that allow individualized assessments of endothelial function to guide the physician, the patient, and the rehabilitative team, are essential. 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 diagnosis of CVD and the guidance of prevention strategies for primary or secondary CVD. From work performed in the current Merit Review Award, the novel single passive leg movement (sPLM)-induced increase in leg blood flow (LBF) approach to assess endothelial function appears to be the clinically relevant tool that has long been missing from the healthcare provider's arsenal. However, the additional studies proposed in this renewal application are needed to confirm this conclusion, especially as it relates to rehabilitation. Importantly, the sPLM paradigm can guide rehabilitation in several ways. First, the high diagnostic value would provide earlier detection of CVD risk, particularly as endothelial dysfunction precedes many of the other risk factors, and the sPLM testing can be used to recommend and motivate patients to undergo rehabilitation. Second, the sPLM assessment can be used to determine the efficacy of rehabilitation in order to guide program development. If rehabilitation is not improving vascular function, the program can be modified to address this CVD risk factor, with the impact on other outcomes in mind. Third, the sPLM paradigm can be used more acutely throughout a rehabilitation program to provide "real-time" feedback about endothelial function, allowing practitioners to personalize the program for each patient, again with other outcomes in mind.

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 our development of the novel sPLM approach to assess endothelial function, fueled predominantly by the current Merit Review Award, 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 weak 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 non-invasive approach for determining vascular function in health and disease. However, predominantly due to several methodological deficiencies and significant challenges in terms of implementation, FMD 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 LNMMA (NG-monomethyl-L-arginine) 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 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 a minimal (0-33%) reduction in FMD after the administration of LNMMA, a NOS blocker, leaving considerable uncertainty as to whether this test can still be relied upon to provide an assay of NO bioavailability. Indeed, our group, using up-to-date measurement techniques and the typical site of assessment (brachial artery), demonstrated 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. Critically, 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. Additionally, changes in FMD are highly dependent upon assessing very small changes in artery diameter. Furthermore, FMD transiently increases with exercise training over the initial 4 weeks, but returns to pre-training values by the 8th week due to arterial remodeling. This plasticity in diameter greatly limits the use of FMD as a diagnostic assessment in clinical and rehabilitation arenas. Finally, although FMD has been demonstrated to correlate with CVD risk, it provides only marginal diagnostic value for CVD. 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 and NO bioavailability.

sPLM as a test of NO bioavailability: Originally, our group and others focused on the peripheral and central hemodynamic responses to sPLM, and its previous variants, 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 LBF. Through various experimental approaches we have characterized the roles of afferent feedback, the muscle pump, perfusion pressure, cardiac innervation/denervation, and age in the sPLM response. However, most germane to this Merit Review renewal application, funded by the current Merit Review Award, we have determined the role NO plays in the sPLM-induced blood flow response in both young and old subjects. 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 sPLM-induced hyperemia, which does not evoke a peripheral metabolic response, is intuitively more likely to be predominantly NO-mediated. Indeed, in young healthy subjects the inhibition of NOS, with an intra-arterial L-NMMA infusion in the passively moved leg, revealed that NO contributes to 65-80% of the hyperemia and vasodilation associated with sPLM. In contrast, there was minimal effect of L-NMMA in the old. The sPLM response is also markedly attenuated in old, compared to young, subjects. Substantial evidence supports NO bioavailability is low in old subjects and, as sPLM is highly NO-mediated, both the diminished sPLM response and diminished effect of L-NMMA are consistent with such diminished NO bioavailability. This supports the sPLM as a good assessment of endothelial function because it tracks physiological differences across populations. This robust and easily measured reduction in NO-mediated vasodilation and subsequent hyperemia suggests that sPLM induced blood flow has significant promise as a new approach to assess NO-mediated endothelial function, an important predictor of CVD risk, and could be an important tool to assess rehabilitation.