The endothelial tissue is the inner lining of the lumen of blood vessels. Till recently it was considered as a tissue which acted as a barrier for blood and its vessels. Recently, it was discovered that apart from acting a barrier it has important role in regulation of blood pressure and other cardiovascular functions. It serves as a major regulator of cardiovascular functioning. The functions of the vascular endothelium, which serves as an autocrine and paracrine organ, include the maintenance of vascular homeostasis by modulating vascular tone, the inhibition of platelet aggregation, the modulation of leukocyte tethering, the regulation of smooth muscle cell proliferation and the modulation of vascular wall permeability. Its dysfunction leads to many diseases and is commonly associated with impaired endothelium-mediated vasodilatation. It is thus, very important to assess the function of endothelial cells. Endothelial dysfunction can result from disease processes, as occurs in septic shock, hypertension, hypercholesterolaemia, diabetes as well as from environmental factors, such as from smoking tobacco products. Various techniques are useful in assessing the endothelial function. Flow-mediated vasodilation (FMD) in the brachial artery measured by ultrasound, and the increase in forearm blood flow (FBF) induced by local infusion of a muscarinic-receptor agonist have both frequently been used to evaluate endothelium-dependent vasodilation (EDV) in the human forearm. In this review an insight has been provided on the techniques used in assessment of the endothelial function, their advantages and disadvantages.
Endothelial cells are simple squamous cells that line the entire circulatory system - the interior of all blood vessels including arteries and veins (as well as the innermost lining of the heart and lymphatics). They possess vasodilator function, as well as anti-thrombotic and anti-inflammatory functions. A key feature of endothelial dysfunction is the inability of arteries and arterioles to dilate fully in response to an appropriate stimulus. Endothelial cells synthesize at least three different vasodilator factors: Nitric oxide, Prostacyclin (PGI2), and an unidentified Endothelium-Derived Hyperpolarizing Factor (EDHF). Heme proteins from blood can also stimulate the inducible isoform of Heme oxygenase (HO-1 also known as heat shock protein 32) which produces carbon monoxide that can relax smooth muscle. Under several pathological conditions, endothelial cells also synthesize several vasoconstricting factors (EDCF), including endothelin, superoxide and vasoconstrictor prostaglandin.
The main vasodilator substance produced by the endothelium is the Endothelium-Derived Relaxing Factor (EDRF), nitric oxide (NO) It was also discovered that NO, apart from its vasodilatory properties, also influenced platelet aggregation, leukocyte adhesion to the endothelium, as well as regulating proliferation of different cell types in the vascular wall. As acetylcholine was known to evoke a profound vasodilation that could be blocked by false analogues of L-arginine, the precursor of NO, this muscarinic-receptor agonist was used to induce vasodilation in the coronary arteries during angiographic procedures and the degree of vasodilation was taken as an index of NO formation.
Endothelial dysfunction is an important factor in many cardiovascular diseases, and is commonly associated with impaired endothelium-mediated vasodilatation. Information about the mechanisms behind this dysfunction has come largely from animal studies or, in humans, through invasive techniques that are not specific to one vascular bed. The central role of the endothelium in vascular regulation is highlighted by the many pathological conditions in which its dysfunction is an important feature. Hypertension chronic heart failure, diabetes, hypercholesterolaemia and generalized atherosclerosis, for example, are all associated with impaired endothelium-mediated vasodilatation. Great emphasis has recently been placed on the role of endothelial dysfunction in the pathogenesis of a wide spectrum of cardiovascular diseases such as atherosclerosis and hypertension.
Animal studies have provided substantial information about the mechanisms involved in the development of vascular disease, but it is also important to investigate these mechanisms directly in humans. Methods using isolated organ preparations have proved valuable in animal models, but the results cannot necessarily be extrapolated directly to humans, and these methods are not appropriate for human subjects. Ideally endothelial function should instead be assessed and, as far as possible, non-invasively.
Recent methodological developments for measuring endothelial microvascular function enable their use as surrogate endpoints in clinical trials.
Endothelial function in humans
Endothelial function can be assessed in freshly isolated blood vessels from humans. Bradykinin caused greater relaxation in the large arteries and resistant small mesenteric arteries, compared to acetylcholine, in one study of Endothelium-Dependent Relaxation (EDR). The bradykinin-induced EDR in the large arteries was due to the combined effect of NO and EDHF, as shown by one-half being sensitive to L-arginine and one half to KCl in the presence of indomethacin and L-arginine. However, in the microvessels bradykinin-induced EDR was insensitive to L-arginine and highly sensitive to KCl in the presence of indomethacin and L-arginine, indicating the relaxation was largely mediated by EDHF but not NO.
In vivo non-invasive assessment: Ultrasound is used to measure flow-mediated dilatation for non-invasive in vivo analysis, and is particularly useful in the clinical setting. By continuously measuring the brachial artery diameter change in response to occlusion and reflow, as demonstrated in Figure 1, the flow-mediated EDR can be assessed. The disadvantage of this technique is that the relation between brachial artery response and coronary artery response is unclear. There is ongoing controversy about whether this brachial artery response truly represents the coronary artery response, and more studies are needed.
Fig.1 :Schematic representation of laser Doppler flowmetry
Although reproducible, this technique is used only by specialists as it involves the cathetering of the brachial artery. Conversely, microvascular function can be studied routinely in humans by using non-invasive Laser Doppler flowmetry of the skin.
Using laser Doppler to investigate skin microvascular reactivity
Laser Doppler is based on the reflection of a beam of laser light. Light undergoes changes in wavelength (Doppler shift) when it hits moving blood cells. The magnitude and frequency distribution of these changes in wavelength are related to the number and velocity of blood cells. Several different signals can be recorded but the red blood cell flux (i.e. the product of the velocity and concentration of moving blood cells within the measuring volume) is used most often. Laser Doppler flowmetry enables the evaluation of cutaneous microvascular blood flow over time.
The major advantage of this technique is its sensitivity at detecting and quantifying relative changes in skin blood flow in response to a given stimulus.
Postocclusive hyperemia
Postocclusive skin reactive hyperemia refers to the increase in skin blood flow above baseline levels following the release of a brief arterial occlusion. It is also called postischemic or reactive hyperemia. It can be characterized by an initial peak in flux that occurs within a few seconds of removal of the occlusion, and a sustained hyperemia. This test is performed by placing a cuff on the distal part of the upper arm and increasing the pressure to 50 mm Hg above the systolic blood pressure. Laser Doppler probes are ideally located on the internal face of the forearm, on skin without dermatological lesions. Many parameters can be quantified from the flux response. The most commonly used as the primary endpoint is peak hyperemia.
Local thermal hyperemia
Local thermal hyperemia leads to a temperature-dependent sustained increase in skin cutaneous flow and achieves a maximal vasodilatation between 42.8 oC and 44.8 oC. Consistent data show that thermal hyperemia is impaired in diabetes. As global tools with which to assess microvascular function, both the axon reflex and the late plateau are impaired in systemic sclerosis. By contrast, patients with low-flow postural tachycardia syndrome exclusively exhibit a decreased plateau, whereas those with chronic spinal cord injury exhibit a decreased axon reflex.
Acetylcholine iontophoresis
Iontophoresis is based on the principle that a charged drug in solution will migrate across the skin under the influence of a direct low-intensity electric current. When combined with Laser Doppler Flowmetry or perfusion imaging, this method enables the detection of alterations in cutaneous blood flow in response to the time-controlled delivery of the vasoactive drug to a patch of skin.
The variability of the responses to ACh was relatively low, both within and between individuals, conforming the suitability of this protocol for experimental assessment of endothelial function in vivo.
Limitations and issues of laser Doppler techniques
The measurement of cutaneous blood flux rather than blood flow: One major limitation of laser Doppler flowmetry is that it is not possible to measure absolute perfusion values (i.e. cutaneous blood flow in ml/min relative to the volume or
weight of tissue).
How to express laser Doppler flowmetry values: Vascular responses to most interventions are standardized to the baseline resting flux level, similar to the flowmediated dilatation of the brachial artery. However, because skin circulation is a vital aspect of normal thermoregulation in humans, variations in ambient and/or local temperature lead to huge differences in cutaneous vascular flow, which are largest in the extremities, where arteriovenous shunts are present
The biological zero: Flux does not reach the value of zero when perfusion is absent. Brownian motion of macromolecules arising from the interstitial space contributes to the remaining signal when red blood cell flow is absent. Although this phenomenon requires that the biological zero be subtracted from flux values expressed as absolute values, it is less crucial when flux values are expressed as a percentage of a standard comparator.
Reproducibility: Laser Doppler flowmetry has often been considered poorly reproducible. However, the major source of variation is the site of measurement. When the recording site is standardized, the day-to-day reproducibility of postocclusive hyperemia, thermal hyperemia and acetylcholine iontophoresis (expressed as absolute values) compares well with that of flow-mediated dilatation of the brachial artery, with each having a coefficient of variation <10%.
In vivo invasive assessment
Invasive in vivo analyses use - 1) venous plethysmography of forearm circulation, 2) quantitative angiography (QCA) and Doppler flow wire assessment of coronary circulation.
An accurate evaluation of endothelial function cab be obtained with these techniques. Continuous monitoring of the changes in diameter in large coronary arteries cab be obtained by combining QCA and Doppler flow wire. Thus both large epicardial coronary artery endothelial function and microvascular endothelial function can be assessed.
Biochemical markers
Several biochemical markers could be used as an indicator of endothelial function: NO plasma level, prostacyclin, thrombomodulin von Willebrand factor, and tissue factor pathway inhibitor, among others. A combination of these markers may help to assess endothelial function systemically.
Table 1: Circulating biomarkers of endothelial function
Vasodilators: Nitrites and nitrates (nitric oxide metabolites), 6-keto PGF1a (prostacyclin metabolite)
Vasoconstrictors : Endothelin-1, Angiotensin II, Thromboxane A2, radical oxygen species, prostaglandin H2
Markers of activation and inflammation : C-reactive protein, cellular adhesion molecules (P- and E-selectin, intercellular adhesion molecule, vascular cell adhesion molecule), monocyte chemoattractant protein-1, interleukins (IL-6, IL-8), tumor necrosis factor-alpha
Markers of hemostasis : Tissue plasminogen activator, plasminogen activator inhibitor-1, von Willebrand factor, thrombomodulin
Others : Asymmetric dimethylarginine, endothelial microparticles, vascular endothelial growth factors, endothelial progenitor cells, circulating endothelial cells
Lp-PLA2 is independently associated with coronary artery endothelial dysfunction and is a strong predictor of endothelial dysfunction in humans.
The advantage of these markers is their clinical simplicity as they quantify, by use of current immunological techniques, circulating factors released by the vascular endothelium and/or are involved in the regulation of its activity. However, this approach can sometimes require delicate conditions of sampling and/or analysis.
Different methods are now available for the clinical assessment of endothelial function that provide complementary information. However, functional testing using pharmacological stimuli appear more specific for the study of resistance arteries. These methods could be associated with the study of conduit arteries endothelial function to enable a selective and comprehensive approach of the heterogeneity of endothelial function in pathophysiology. These different approaches are actually used in research but their development should allow their use for early diagnosis and therapeutic management in clinical practice as they demonstrated the major ability of endothelial dysfunction to predict cardiovascular events and although additional interventional studies are necessary to confirm the correlation between improvement of endothelial function and cardiovascular outcome.
(The authors AN Nagappa, Kunal Saxena, Ruchir Bhomia, Apurvasena Parikh are with Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104. and Ranjitha Inguva is with Department of Pharmacy, Birla Institute of Technology and Sciences (BITS), Pilani).