Vascular endothelium is a cellular layer lining the inner part of blood vessels, including arteries and veins. Endothelium is presently looked upon as an important metabolically active autocrine/paracrine/endocrine organ that regulates cardiovascular function and maintains vascular homeostasis by: modulating vascular tone; regulating solute transport into cell components of the vessel wall, local cellular growth, and extracellular matrix deposition; protecting the vessel from the potentially injurious consequences of substances and cells circulating in blood; and regulating the hemostatic, inflammatory, and reparative responses to local injury. One of the main functions of endothelium is to produce or release substances, such as nitric oxide (NO), that control the behaviour of the blood vessels such as their dimensions, elasticity, permeability and reactivity, including the ability to constrict and dilate. Endothelium-derived mediators regulate not only blood flow and permeability vascular elasticity, reactivity and structure, but also local and systemic inflammatory response as well as thromboresistance of vessels. Vasoprotective endothelial mediators such as nitric oxide (NO), prostacyclin (PGI2) endothelium-derived hyperpolarising factor (EDHF), bradykinin (Bk) tissue plasminogen activator (t-PA), thrombomodulin (TM) or ADP-ase do exert antithrombotic, anti-inflammatory and vasoprotective action.
On the other hand, excessive production by endothelium of superoxide anions (O2-), isoprostanes, angiotensin II (ang II), endothelin 1 (ET-1), plasminogen activator inhibitor (PAI-1), tissue factor (TF), von Willebrandt factor (vWF), chemokines (e.g. monocyte chemotactive protein MCP-1), cytokines (e.g. IL-6), and increased expression of adhesion molecules (e.g. selectin P, ICAM-1) promote inflammation and thrombosis of vascular wall that may eventually lead to the development of atherosclerotic lesion. Accordingly, healthy endothelium is essential for undisturbed functioning of the cardiovascular system, while endothelial dysfunction leads to its various pathologies. In particular, endothelial dysfunction is pivotal to atherogenesis, it is present at the earliest stages (e.g. preceding angiographic or ultrasonic evidence of obstructive plaque) as well as later stages of arterial disease, contributing to clinical sequelae related to tissue damage (eg, ischemia, infarction, and organ failure).
Endothelial dysfunction in most general terms refers to an impairment of the ability of the endothelial cell layer to produce an appropriate vasodilatory response to stimuli. Many studies provided evidence that endothelial dysfunction (assessed on the basis of the impairment of NO-dependent vasodilatation) may be regarded as prognostic factor for the development of adverse cardiovascular events. Indeed, relative risk for adverse outcomes is elevated approximately 10-fold when there is evidence of coronary or peripheral endothelial dysfunction.
Various conditions, including hypercholesterolemia, systemic hypertension, smoking, diabetes, congestive heart failure, pulmonary hypertension, estrogen deficiency, hyperhomocysteinemia, and the aging process itself, have been associated with impaired function (dysfunction) of endothelium. As a result, the vessel wall in these conditions may promote inflammation, oxidation of lipoproteins, smooth muscle proliferation, extracellular matrix deposition or lysis, accumulation of lipid-rich material, platelet activation, and thrombus formation. All of these consequences of endothelial dysfunction may contribute to development and clinical expression of atherosclerosis. The potential consequences of endothelial dysfunction further include coronary constriction or inadequate dilation during physical or mental stress, producing myocardial ischemia; plaque rupture and thrombosis, causing unstable angina or myocardial infarction; and reperfusion injury after thrombolysis.
Several methods and apparatuses for non-invasive evaluation of the health of vascular endothelium in vivo have been developed.
In particular, methods are known that are based on monitoring the physiological conditions or characteristics of the arteries in the patient's limb after reactive hyperemia.
Reactive hyperemia is a physiological phenomenon that occurs in a patient after blocking (or occlusion) of a major artery. Such blocking or occlusion of artery in the limb, such as brachial artery, is typically done by inflating a blood pressure cuff slightly above systolic pressure for a period of about 5 minutes. Anoxia or severe hypoxia in the limb downstream from the occluded artery is usually a result of such blocking. Sudden release of the blocking causes endothelial cells to react by generating NO and dilating. The phenomenon of reactive hyperemia lasts up to 10 minutes before return to pre-test blood volume values. Blood flow is a characteristic of the artery, and under reactive hyperemia blood flow through an artery, vein or limb is significantly greater as compared with normal blood flow.
Currently the most popular method is flow mediated dilatation (FMD), a non-invasive technique based on monitoring of diameter of arteries after reactive hyperemia with a two-dimensional ultrasound and Doppler ultrasound. Its results correlate well with invasive coronary endothelial testing as well as with the presence and severity of coronary atherosclerosis. This technique is described for example in a review by S. Patel. And D. S. Celermajer, Pharmacological Reports 2006, 58, suppl. 3-7. However, this method is quite expensive, requires sophisticated equipment and highly specialized operators, is highly operator dependent and is poorly reproducible due to variability of measurements and poor resolution relative to arterial size. Hence, its use is limited and the method is not applicable on a more general basis.
For the purpose of assessment of vascular endothelial function changes of other physical parameters in response to reactive hyperemia have been also used, such as fingertip skin temperature (WO2005118516; N. Ahmadi et al. Int. J. Cardiovasc. Imaging (2009) 25:725-738), blood pressure in a finger (pulse wave amplitude) using plethysmography (EP1360929, EP1992282, WO00/57776, EP2110074), and peripheral arterial tone (WO2000/074551, WO2002/034105).
Non-invasive technique for detection of endothelial dysfunction based on monitoring blood flow related changes in the level of a substance present in a limb after reactive hyperemia is disclosed in WO03/051193. The method involves blocking blood flow in the limb to stimulate endothelial function and then releasing the blood flow block to observe, measure and record said changes as a function of time, said changes being indicative of endothelial dysfunction. Said substance can be a tracer substance injected in a vein, such as a radiation emitter or a contrast agent, and the ingress of said tracer into the limb is detected and measured, for example by means of gamma ray detection. Tracer measurement in a pair of two laterally opposed limbs should be performed and the tracer presence compared between both limbs. Alternatively, a physical characteristics of the limb, such as temperature or color, or a property of a metabolic or other biochemical product circulating in the limb following the release of the blood flow block, such as O2, CO2 or reduced hemoglobin, is measured by a suitable technique. As suitable techniques there were suggested gas emissions across the skin surface within a cell placed on the skin surface, optical techniques, such as spectral analyzers or optical transmission/diffusion detectors, such as the visible-reflectance hyperspectral analysis, and EPR/NMR techniques. Either the appearance rate of a depleted substance or the disappearance (depletion) of an accumulated product can be detected. A rate of change of the measured parameter shortly after release of the occlusion or blockage is suggested as a primary factor in determining endothelial dysfunction. In the case of the use of a tracer, the rate for both the blocked limb and the contra-lateral control limb is measured.
It has been established that endothelial dysfunction is an early event and major risk factor for atherosclerosis and an important indicator for a medical professional, allowing for early diagnosis of the risk of cardiovascular disease.
Testing endothelial function is therefore a highly desirable alternative for a diagnostic approach based on performing a set of various biochemical tests, especially in apparently healthy individuals, i.e. individuals not showing any signs of cardiovascular disease.
A testing method is needed that would allow to evaluate function and detect any dysfunctions at an early stage of impairment in order to identify patients for prophylactic or therapeutic intervention to improve the dysfunction and/or for further more detailed and complicated diagnostic tests.
The need exists to provide a non-invasive test for evaluating endothelial function which would be reliable, easy to carry out and inexpensive, and thus applicable for tests in large patient populations, for example for screening purposes.
There is also a need for a simple, quick and non-expensive test that would allow to monitor and control the response of a patient to a medical treatment of cardiovascular disease.