Index
Most animal epithelial cells have a fluff-like coating on the outer surface of their plasma membranes.
Glycocalyx, also known as the pericellular matrix, is a covering of glycoprotein and glycolipids that surrounds the cell membranes of some bacteria, epithelia, and other cells.
This coating consists of various carbohydrate moieties of membrane glycolipids and glycoproteins, which serve as backbone molecules for support.
In general, the carbohydrate portion of glycolipids found on the surface of plasma membranes helps these molecules contribute to cell cell recognition, communication, and intercellular adhesion.
Glycocalyx is a type of identifier that the body uses to distinguish between its own healthy cells and transplanted tissues, diseased cells, or invading organisms.
Included in the glycocalyx are cell adhesion molecules that allow cells to adhere to each other and guide the movement of cells during embryonic development.
Glycocalyx plays a major role in the regulation of vascular endothelial tissue, including modulation of the volume of red blood cells in capillaries.
Silt on the outside of a fish is an example of glycocalyx. The term was initially applied to the polysaccharide matrix that lines epithelial cells, but its functions have been found to go much further.
History
The endothelial glycocalyx was already visualized some 40 years ago by Luft using electron microscopy. Still, relatively little is known about the composition and function of this layer.
In recent decades, it has been increasingly appreciated as an important factor in vascular physiology and pathology, as described in 2000 in a review by Pries et al. and in other more recent reviews.
Interest in the physiological role (patho) of glycocalyx began with the observation of the low and variable capillary tube hematocrit, which depended on the level of metabolic and pharmacological activation of the vascular system.
The relationship between metabolism and agonist-induced increases in red blood cell velocity, on the one hand, and hematocrit, on the other, could be partly explained by plasma skimming as a direct continuation of the Fåhraeus effect.
However, this relationship is dissociated from local treatment of the microvessels with heparinase, an enzyme that breaks down heparan sulfates in glycocalyx.
This finding was in agreement with theoretical estimates predicting a 1.2 μm slow-moving plasma layer on the endothelium.
In vivo studies have revealed that the glycocalyx in muscle capillaries is a layer approximately 0.5 μm thick, which covers endothelial cells and determines the luminal domains for macromolecules, red and white blood cells.
More recent studies indicate that the thickness of the glycocalyx increases with the vascular diameter, at least in the arterial system, which varies from 2 to 3 μm in small arteries to 4.5 μm in carotid arteries.
To date, many studies indicate a variety of physiological (patho) functions for endothelial glycocalyx; In addition to modulating the capillary filling of red blood cells, glycocalyx can affect many other functions (dis) of the vascular system.
While the vascular endothelium is now believed to be actively involved “in every pathology that presents vascular projections,” the same saying may well be true for glycocalyx.
Assessing this possible involvement of endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge.
Composition
Endothelial glycocalyx is a carbohydrate-rich layer that lines the vascular endothelium. It is considered to be connected to the endothelium through several “major” molecules, primarily proteoglycans and also glycoproteins.
These form a network into which soluble molecules derived from plasma or endothelium are incorporated.
More luminally, glycocalyx is made up of soluble components of plasma, linked together directly or by soluble proteoglycans and / or glycosaminoglycans.
There is a dynamic balance between this layer of soluble components and the flowing blood, continually affecting the composition and thickness of the glycocalyx.
In addition, glycocalyx suffers from enzymatic or shear-induced shedding. The dynamic balance between biosynthesis and shedding makes it difficult to define glycocalyx geometrically.
The mesh composition of membrane-bound proteoglycans, glycoproteins and glycosaminoglycans and the composition of associated plasma proteins and soluble glycosaminoglycans cannot be viewed as a static image.
Instead, the layer as a whole, also known as the endothelial surface layer (ESL), is highly dynamic, with membrane-bound molecules constantly being replaced and without a defined boundary between locally synthesized elements and associates.
Membrane-bound hyaluronan can reach lengths of> 1 μm.
Direct visualization techniques do not demonstrate clear compositional differences within the glycocalyx, from the endothelial membrane to the vascular lumen, but indicate that the endothelial glycocalyx resembles an intricate 3D self-assembled network of various polysaccharides.
The enzymatic removal of any of its components dramatically affects the properties of glycocalyx, exemplifying the importance of considering the synergistic interaction of all constituents of glycocalyx as a whole.
Location
In vascular endothelial tissue
Glycocalyx is found on the apical surface of vascular endothelial cells lining the lumen.
When vessels are stained with cationic dyes, such as Alcian blue, transmission electron microscopy shows a small, irregularly shaped layer extending at approximately 50-100 nm in the light of a blood vessel.
Another study used cryotransmission electron microscopy and showed that endothelial glycocalyx could be up to 11 μm thick. It is present in a wide range of microvascular beds (capillaries) and macrovessels (arteries and veins).
Glycocalyx also consists of a wide range of enzymes and proteins that regulate the adhesion of leukocytes and thrombocytes, since its main role in the vasculature is to maintain plasma and vessel wall homeostasis. These enzymes and proteins include:
- Synthesis of endothelial nitric oxide (endothelial SON).
- Extracellular superoxide dismutase (SDE3).
- Angiotensin Converting Enzyme.
- Antitrombina III.
- Lipoprotein lipase.
- Apolipoproteínas.
- Growth factors.
- Chemokines.
The enzymes and proteins listed above serve to strengthen the glycocalyx barrier against vascular and other diseases.
Another main function of glycocalyx within the vascular endothelium is that it protects the vascular walls from direct exposure to blood flow, while serving as a barrier to vascular permeability.
Its protective functions are universal throughout the vascular system, but its relative importance varies depending on its exact location in the vasculature.
In microvascular tissue, glycocalyx serves as a vascular permeability barrier by inhibiting clotting and adhesion of leukocytes.
Leukocytes must not adhere to the vascular wall because they are important components of the immune system that must be able to travel to a specific region of the body when necessary.
In arterial vascular tissue, glycocalyx also inhibits leukocyte clotting and adhesion, but through mediation of shear stress-induced nitric oxide release.
Another protective function throughout the cardiovascular system is its ability to affect interstitial fluid filtration from capillaries into the interstitial space.
Glycocalyx, found on the apical surface of endothelial cells, is composed of a negatively charged network of proteoglycans, glycoproteins, and glycolipids.
In bacteria and nature
A glycocalyx, which literally means “sugar coat”, is a network of polysaccharides that project from the cell surfaces of bacteria, which classifies it as a universal surface component of a bacterial cell, located just outside the bacterial wall. mobile.
A distinct gelatinous glycocalyx is called a capsule, while a diffuse, irregular layer is called a slime layer. This coat is extremely hydrated and is dyed ruthenium red.
Bacteria that grow in natural ecosystems, such as in the soil, bovine intestines or the human urinary tract, are surrounded by a kind of microcolony enclosed in glycocalyx.
It serves to protect the bacteria from harmful phagocytes by creating capsules or allowing the bacteria to adhere to inert surfaces, such as teeth or rocks, through biofilms.
For example, Streptococcus pneumoniae attaches itself to lung cells, prokaryotes or other bacteria that can fuse their glycocalices to envelop the colony.
In the digestive tract
A glycocalyx can also be found in the apical part of the microvilli within the digestive tract, especially within the small intestine.
It creates a 0.3 μm thick network and consists of acidic mucopolysaccharides and glycoproteins that project from the apical plasma membrane of absorbent epithelial cells.
It provides an additional surface for adsorption and includes enzymes secreted by the absorption cells that are essential for the final steps of protein and sugar digestion.
Other generalized functions
Protection : cushions the plasma membrane and protects it from chemical damage.
Immunity to infection : allows the immune system to selectively recognize and attack foreign organisms.
Cancer defense : Changes in the glycocalyx of cancer cells allow the immune system to recognize and destroy them.
Transplant Compatibility – forms the basis for compatibility of blood transfusions, tissue grafts, and organ transplants.
Cell adhesion : unites cells so that tissues do not fall apart.
Inflammation regulation : Glycocalyx coating on endothelial walls of blood vessels prevents leukocytes from rolling or coalescing into healthy states.
Fertilization : allows the sperm to recognize and bind to the eggs.
Embryonic development : directs embryonic cells to their destinations in the body.
Visualization techniques
Due to the functional importance of the endothelial glycocalyx, the development of direct visualization techniques is crucial to establish its exact function.
Glycocalyx can be labeled by administering specific markers that bind to one or more of its components, making them fluorescent or detectable.
The preparation of (parts of) the vessel would then make it possible to obtain specific microscopic images of the endothelial glycocalyx.
Unfortunately, glycocalyx is very vulnerable and is easily disturbed or dehydrated during vessel handling and preparation protocols.
As a result, the dimensions of the glycocalyx are easily underestimated, which is illustrated by the first images of the glycocalyx, made by transmission electron microscopy (MET) in 1966 using the ruthenium red probe.
The glycocalyx thickness measured in this way approached 20 nm in the capillaries. Since then, many other attempts have been made to image the glycocalyx using transmission electron microscopy.
In bovine aortic endothelial cells under 3.0 Pa shear conditions, the glycocalyx was reported to be 40 nm thick.
These dimensions did not meet the theoretical estimates that predict glycocalyx to be up to 1 μm thick.
Using a new staining protocol with Alcian blue 8GX, van den Berg et al. Transmission electron microscopy was recently applied to measure the dimensions of the endothelial glycocalyx in rat myocardial capillaries.
This study revealed that endothelial cells are covered by a glycocalyx 200 to 500 nm thick.
Hyaluronidase treatment prior to fixation and staining resulted in a significant reduction of this layer at 100-200 nm.
The Haraldsson and Rostgaard and Qvortrup groups improved the transmission electron microscopy staining protocol using fluorocarbon-based oxygen fixatives, revealing glycocalyces as thick as 60-200 nm in glomerular capillaries and 50-100 nm in intestinal fenestrated capillaries. .
The new staining and preparation protocols apparently improved glycocalyx preservation in transmission electron microscopy experiments. However, transmission electron microscopy cannot be used in the in vivo situation.
About 30 years after the first transmission electron microscopy images were made, Vink et al. used intravital microscopy to visualize endothelial glycocalyx in hamster cremaster muscle capillaries in vivo using indirect approaches.
Glycocalyx was recognized as an “exclusion zone” or “gap” between the flowing red blood cells and the endothelium. Furthermore, the plasma was labeled with a fluorescent dextran, and the glycocalyx then appeared as a plasma exclusion zone.
Interestingly, no exclusion zone was found for white blood cells, suggesting that they have the ability to compress glycocalyx in these vessels, complying with the estimated low stiffness of glycocalyx.
Subtraction of the diameter of the plasma column from the anatomical internal diameter revealed the dimensions of the glycocalyx, which appeared to be 0.4-0.5 μm thick.
This method has been used in many studies since then, mainly in the microcirculation of the cremaster muscle of hamsters or mice.
This tissue is suitable for intravital microscopy because it is thin and translucent, allowing clear visualization of microvascular endothelial cells and blood cells, with low or absent vessel wall movement.
In addition, local flow velocities can be measured. However, estimation of the thickness of the glycocalyx using methods based on intravital microscopy is indirect. Furthermore, intravital microscopy cannot be applied to image endothelial glycocalyx in larger vessels.
Direct visualization of glycocalyx has been accomplished through various approaches, most using lectins which are proteins that bind to specific disaccharide residues of glycosaminoglycan chains.
Other tags include antibodies to heparan sulfate, syndecane-1, or hyaluronan. By attaching these markers to a fluorescent probe, advanced microscopic techniques can be applied to visualize the glycocalyx.
Confocal laser scanning microscopy (CLSM) enables optical sectioning with good optical resolution, allowing 3D reconstruction of the sample.
Lectin-labeling of the glycocalyx of cultured human umbilical vein endothelial cells and subsequent confocal laser scanning microscopy imaging revealed a surface layer as thick as 2.5 ± 0.5 µm.
Confocal laser scanning microscopy has also been used to detect changes in the concentration of fluorescently labeled lectins in the glycocalyx of fixed rat mesentery postcapillary venules in ischemia / reperfusion and inflammation.
Because larger vessels have thicker walls, resulting in shallower depths of light penetration with significant loss of resolution at greater depths (> 40 μm).
Due to greater signal dispersion, confocal laser scanning microscopy is less suitable for imaging the glycocalyx in arteries.
A promising technique for directly visualizing glycocalyx in larger vessels, both ex vivo and in vivo, is two-photon laser scanning microscopy (TPLSM).
Two-photon laser scanning microscopy relies on the excitation of a fluorophore by simultaneous uptake (i.e., within 10-18 s) of two red photons, rather than one blue photon as in conventional fluorescence excitation.
The use of long wavelength red photons reduces scattering and thus increases the depth of penetration into the tissue.
The excitation of the fluorophore and the consequent fluorescence only occurs at the focal point of the illumination cone, since the probability of excitation of two photons depends on the intensity squared of the excitatory photons.
Any light received by the photomultipliers has to originate from the focal position, so the scattering of the emitted photons does not influence the resolution and no pitting is required.
As a consequence, the two-photon laser scanning microscope offers good resolution and optical cross-section at a reasonable acquisition speed, while the bleaching and phototoxicity of the dyes are limited to the focal position.
The combination of improved penetration depth, good resolution, optical cutting, and low phototoxicity makes two-photon laser scanning microscopy a suitable technique for visualizing the delicate endothelial glycocalyx in larger vessels.
This idea was confirmed by a recent study by Megens and colleagues in which endothelial glycocalyx was imaged with two-photon laser scanning microscopy in intact mouse carotid arteries; the thickness of the glycocalyx was 4.5 ± 1.0 μm.
As the two-photon laser scanning microscope is also applied in vivo in rodents, it could be a good approach for the in vivo visualization of glycocalyx in the macrocirculation of these animals.
Conclusions
On the vascular endothelium, the glycocalyx is a membrane-bound mesh in which plasma-derived molecules are integrated.
It performs a variety of functions, important in normal vascular physiology and also in vascular disease.
Although data from experiments in microcirculation and, more recently, macrocirculation strongly suggest a vasculoprotective role for glycocalyx, research on this topic is hampered by the lack of good visualization technique.
Two-photon laser microscopy can be a successful tool to achieve direct visualization of glycocalyx in larger arteries in rodents, both ex vivo and in vivo, with the possibility of analyzing focal variations in the composition or integrity of this layer.
