This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium-and plasmaderived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cellvessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro-and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Velocity profiles were determined in rabbit mesenteric arterioles (diameter 17-32 /xm). A good spatial resolution was obtained by using the blood platelets as small and natural markers of flow, providing for the first time in vivo detailed, quantitative information about the shape of the velocity profiles in micro vessels. In some experiments red blood cell velocity profiles were recorded as well. Easy detection of the cells of interest could be achieved by labelling them selectively with a fluorescent dye and visualizing them by intravital fluorescence video microscopy, using flashed illumination. Pairs of flashes were given with a short, preset time interval between both flashes, yielding in one TV picture two images of the same cell displaced over a certain distance for the given time interval. Velocity and mean radial position of cells, flowing within an optical section around the median plane of the vessel, were determined. The shape of the velocity profiles of platelets and red blood cells was similar. The profiles were flattened as compared to a parabola, both in systole and diastole. Vessel diameter did not change measurably during the cardiac cycle. As an index of the degree of blunting of the profiles, the ratio of the maximal and mean velocity of the profile was used, which is 2 for a parabola and 1 for complete plug flow. The index ranged from 1.39 to 1.54 (median 1.50), and increased with vessel diameter. Calculations showed that the blunting of the profiles cannot be explained by an influence of the finite depth of the optical section. (Circulation Research 1986;59:505-514) knowledge of the velocity profile in microvessels, i.e., the velocity distribution over the cross-sectional area of these vessels, is important for several reasons. First, adequate description of blood flow through small vessels requires information about the velocity gradients or shear rates in the fluid.1 Second, transport of cellular components in the blood is determined by both their distribution over the cross-sectional area of the vessel and the velocity profile. For instance, knowledge of the distribution of blood platelets in microvessels 2 and their velocity profile allows the calculation of the rate of platelet delivery in hemostatic plug or thrombus formation in these vessels. Third, to estimate volume flow in microvessels photometric methods are widely used, 3 employing an empirical factor derived from a model in which a parabolic velocity profile is assumed. However, in this approach an error will be made if in vivo the velocity profiles are more flattened.In vitro studies in glass tubes on the velocity profiles of ghost cell suspensions 5 Until now, precise measurement of velocity profiles in small blood vessels has not been performed in vivo for technical reasons. Photometric methods cannot be used to determine a profile because the system does not provide a direct measure of the red blood cell velocity in the plane of sharp focus. 34 With high-speed cinematography, displacement of red blood cells can be followe...

Background-Low birth weight is associated with an increased incidence of cardiovascular diseases, including hypertension, later in life. This suggests that antenatal insults program for fetal adaptations of the circulatory system. In the present study, we evaluated the effects of mild hypoxia on cardiac function, blood pressure control, and arterial structure and function in near-term chick embryos. Methods and Results-Chick embryos were incubated under normoxic (21% O 2 ) or hypoxic (15% O 2 ) conditions and evaluated at incubation day 19 by use of histological techniques, isolated heart preparations, and in vivo measurements of sympathetic arterial tone and systemic hemodynamics. Chronic hypoxia caused a 33% increase in mortality and an 11% reduction in body weight in surviving embryos. The lumen of the ascending aorta in hypoxic embryos was 23% smaller. Left ventricular systolic pressure was 22% lower, and heart weight/body weight ratio was 14% higher. In resistance arteries of hypoxic embryos, in vivo baseline tone was 23% higher, norepinephrine sensitivity was similar, and norepinephrine release from sympathetic nerves increased 2-fold, indicating sympathetic hyperinnervation. Mean arterial pressure and heart rate were similar under resting conditions, but chronically hypoxic embryos failed to maintain blood pressure during acute stress. Conclusions-This study indicates that mild hypoxia during embryonic development induces alterations in cardiac and vascular function and structure and affects hemodynamic regulation. These findings reveal that antenatal insults have profound effects on the control and design of the circulatory system that are already established at birth and may program for hypertension and heart failure at a later age.

Background: Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study (sub)cellular details in arteries with wall thicknesses above 50 µm. Methods: Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries. Results: In thin slices, (sub)cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E^–/– mice demonstrated that in contrast to confocal or wide-field microscopy, TPLSM can be used to visualize (sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E^–/– mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining. Conclusions: Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse.