Significance of the endothelial glycocalyx

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The appearance of the endothelial glycocalyx in Question 18 from the second paper of 2014 demonstrates that this topic has high penetrance and popularity among the college examiners.

For a more thorough overview, the pathologically curious reader is directed to Weinbaum et al (2007)- The Structure and Function of the Endothelial Glycocalyx Layer. Additionally, there is an entire website devoted to the activities of a glycocalyx research team, which features professional-looking diagrams and a list of recent publications.

For the rest of us, a brief summary will suffice. Owing to certain cognitive defects on the part of the author, the summary offered below can hardly be described as brief. Fortunately, a brilliant LITFL CCC alternative waits to rescue the time-poor exam candidate. In point form, its content can be compressed as follows:

Constitutents of the endothelial glycocalyx

• The glycocalyx is a thin (500-2000nm) hydrated gel-like layer on the luminal surface of the vascular endothelium.
  • Given that the vascular endothelium covers a total surface of around 4000-7000m², in spite of its microscopic thinness this layer actually occupies about 25% of the total intravascular volume.
• The name means "sweet husk", referring to the high polysaccharide content.
• It is in fact composed of a vast variety of macromolecules, including:
  • Glycoproteins
  • Polysaccharides
  • Proteoglycans
  • Glycosaminoglycans, such as heparan sulfate, chondroitin sulfate and hyaluronic acid
• These molecules are characterised by a polyanionic charge; this helps repel circulating platelets.
• The exact composition varies greatly according to the local microenvironment
• At any given time, it also contains constintuents of the routine molecular traffic which passes through it or lodges within it, such as:
  • Plasma proteins
  • Enzymes and enzyme inhibitors
  • Growth factors
  • Cytokines
  • Amino acids
  • Cations
  • Water

The glycocalyx is fragile but self-repairing. The passage of a white cell through a tight-fitting capillary can shred it completely, and yet it will restore itself in less than 1 second by adsorbing plasma constituents.

Additionally, it must be mentioned that certain tissues possess capillaries which are intentionally denuded of glycocalyx. Specific examples include the choroid plexus, secretory areas of endocrine glands, hepatic sinusoids and the reticuloendothelial system of the spleen. Functionally, glomerular capillaries also act as if they have no glycocalyx (they actually do have it, but the endothelium there is full of massive fenestrations).

Functions of the glycocalyx

Immunological and barrier functions

• Forms the interface between the vessel wall and moving blood.
• Acts as the exclusion zone between blood cells and the endothelium
• Acts as a barrier against leakage of fluid, proteins and lipids across the vascular wall.
• Interacts dynamically with blood constituents.
• Acts the "molecular sieve" for plasma proteins, forming the basis of the Starling principle and acting as the origin of the oncotic forces which control the transcapillary movement of water.
• Modulates adhesion of inflammatory cells and platelets to the endothelial surface.
• Functions as a sensor and mechanotransducer of the fluid shear forces to which the endothelium is exposed; thus the glycocalyx mediates shear-stress-dependent nitric oxide production.
• Retains protective enzymes (eg. superoxide dismutase).
• Retains anticoagulation factors, eg:
  • Tissue factor inhibitor
  • Protein C
  • Antithrombin

Role of the glycocalyx in transvascular fluid exchange

An excellent BJA review by the Woodcocks (2012) describes in exhausting detal the 2010 Levick and Michel revision to the original Starling model of hydrostatic-oncotic interactions influencing the distribution of volume between the intravascular and extravascular spaces. A more detailed discussion of Starling's Principle is carried out in the Fluid Physiology section; it is a deep rabbit hole and diving into it can be omitted in this discussion as the CICM fellowship exam candidate is already at least dimly aware of the concepts (having presumably passed some sort of primary exam).

In brief, the key glycocalyx-related features of the revised Starling model are as follows:

• The glycocalyx is the semipermeable membrane, not the endothelium
• The oncotic pressure gradient is between the plasma and the subglycocalyx space (not the plasma and the interstitial space).
• While the glycocalyx is intact, the net outward movement of fluid is opposed by, but not reversed, by the oncotic pressure gradient between the plasma and the subglycocalyx space.
• Thus there is no "reabsorption" in the venules as was previously thought.
• The effect of interstitial fluid oncotic pressure on the net microvascular fluid exchange is minimal.
• If the porosity of the glycocalyx is increased by inflammation, and/or if the capillary pressure and blood flow increase, the net movement of fluid out of the capillary increases dramatically.
• This underlies the "leaky capillary" phenomenon seen in inflammation and sepsis.

Dysfunction of the endothelial glycocalyx

Causes of glycocalyceal dysfunction

• Thinning of the glycocalyx layer is caused by generally any pathological process that is known to cause atheroma formation:
  • Hyperglycaemia
  • Hyperlipidaemia
  • Smoking
• Shedding of the glycocalyx layer is caused by
  • Inflammation, mediated by TNFα (this is prevented by hydrocortisone)
    • Other implicated inflammatory mediators listed by this article include C-reactive protein, adenosine (at A₃ adenosine receptors), bradykinin and mast cell tryptase.
  • Ischaemia-reperfusion injury (which halts the endothelial synthesis of glycosaminoglycans)
  • Hypervolemia (mediated by actions of the ANP)
  • Hydroxyethyl starch (actually, its the same study that investigated the effects of hypervolemia - the investigators used HES boluses perioperatively. So was it the volume, or was it the starch itself?)
  • Major vascular surgery (seems like a consequence of bypass and ischaemia/reperfusion)
  • Major abdominal surgery (even without much abdominal sepsis)
  • Sepsis (where it appears to play a fairly central role)

Consequences of having a disrupted glycocalyx

• Local hypercoagulability
• Global autoheparinisation (especially during trauma)
• Increased capillary permeability (demonstrated by some elegant capillary haematocrit studies)
  • Tissue and organ oedema
  • Impaired microcirculatory oxygen distribution
  • Loss of vascular responsiveness
• Increased platelet aggregation, leading to microvascular thrombosis and DIC
• Increased leucocyte-endothelium interaction, leading to inflammation

Importance of glycocalyx in sepsis

A good opinion piece from Critical Care (2012) is available to address this exact issue.

It is, however, somewhat limited in scope. The true pathology nerd will get more from this article on endothelial dysfunction.

In brief summary:

• TNFα-mediated inflammation, inflammatory mediator release and resuscitation hypervolemia (among many other things) lead to glycocalyx degradation.
• Glycocalyx degradation catalyses several simultaneous pathophysiological events:
  • Capillary leak;
    • thus, tissue and organ oedema
    • thus, impaired microcirculatory oxygen distribution
    • thus, impaired organ system function
  • Increased platelet activation and increased local hypercoagulability
    • thus, local microthrombosis with resuting microvascular shunting
      • thus, impaired tissue perfusion and lactic acidosis
    • systemically, this may result in DIC
  • Loss of vascular responsiveness
    • thus, vasoplegia and hypotension

Is there any direct evidence for this?

• Microcirculatory dysfunction in animal models of sepsis correlates with decreased enothelial thickness, suggesting that the glycocalyx is degraded.
• The negative charge of the endothelium has been demonstrated to decrease in animal models of sepsis, which was associated with interstitial oedemam and interpreted as degradation of the glycocalyx.
• Endotoxin infusion results in the appearance of glycocalyx components in the bloodstream, suggesting that the layer is being shed.
• Concentration of glycocalyx components shed into the bloodstream correlates with sepsis severity.

Assessment of glycocalyceal integrity and function

If one takes the glycocalyx seriously, one finds oneself treating it as a neglected organ system.

Therefore, it must have its equivalent of the ECG, and its equivalent of the troponin.

Monitoring glycocalyx integrity directly

• Optical measurement of the distance between the endothelium and the erythrocytes can be used to assess the thickness of the glycocalyx in vivo: the column of erythrocytes moving though the capillary widens as the glycocalyx thins.
  • This was first confirmed by microscopy of the cremaster muscle in a laboratory hamster, and then in 24 healthy males (using orthogonal polarization spectral imaging).

Monitoring glycocalyx integrity indirectly

• Dextran-40 (molecular weight around 40,000 Da) is small enough to penetrate the glycocalyx, and distributes into it when infused into the bloodstream.
• Dextran-70 (molecular weight around 70,000 Da) is too large to penetrate the glycocalyx, and distributes only into the blood volume. Alternatively, one can use labelled erythrocytes.
• Both Dextran species can be infused simultaenously, and their volumes of distribution can be measured.
• The difference between these volumes is therefore (theoretically) an accurate representation of the volume of the glycocalyx.
• Practically, it is impossible to be sure about this tracer dilution method, because nobody knows what the Dextran-40 partition coefficient is between the plasma and the glycocalyx (and how it changes in disease states)

Biomarkers of glycocalyx damage

• Basically, these are components of the glycocalyx, macromolecules which (clearly) don't belong in the bloodstream. Finding these molecules in great number is therefore suggestive that the glycocalyx is being damaged (i.e. it is sloughing off from the endothelium and floating around in the bloodstream)
• Markers used by investigators have included the following molecules:
  • Syndecan-1 (associated with increased mortality in human trauma)- this marker is the most thoroughly investigated, peaks earliest, and seems to be the front runner.
  • Heparan sulfate (porcine model of endotoxin shock) - delayed peak
  • Hyaluronic acid (increases in post-resuscitation syndrome) - delayed peak

Strategies to protect and repair the glycocalyx

A 2014 review of this vascular barrier identified 236 papers and 19 studies interested in its defence.

The well-accepted measures are as follows:

• Corticosteroids
• Normoglycaemia (maintain it)
• Normovolaemia (as in, don't overload them)

There are also experimental treatments and wild speculation:

• Albumin (the evidence is thus far limited to disembodied animal organs in cold solution)
• Polyethylene glycol (as an antioxidant)
• Nitric oxide (also as an antioxidant)
• Adenosine A_2A receptor agonists (antioxidant effect)
• TNF-α inhibitors - specifically etanercept (anti-inflammatory effect)
• Allopurinol (antioxidant)
• Heparin (antioxidant)
• Sulodexide (a mixture of 80% heparan sulfate and 20% dermatan sulfate)
• Hyalouronan (to maybe stimulate the repair of this layer by supplying raw ingredients)
• Chondroitin sulphate (also repair of the layer)
• Antithrombin III (anti-inflammatory effect)
• Lidoflazine (an old-school calcium channel blocker)