Structure and function of the endothelial surface layer: unraveling the nanoarchitecture of biological surfaces

Abstract Among the unsolved mysteries of modern biology is the nature of a lining of blood vessels called the ‘endothelial surface layer’ or ESL. In venous micro-vessels, it is half a micron in thickness. The ESL is 10 times thicker than the endothelial glycocalyx (eGC) at its base, has been presumed to be comprised mainly of water, yet is rigid enough to exclude red blood cells. How is this possible? Developments in physical chemistry suggest that the venous ESL is actually comprised of nanobubbles of CO2, generated from tissue metabolism, in a foam nucleated in the eGC. For arteries, the ESL is dominated by nanobubbles of O2 and N2 from inspired air. The bubbles of the foam are separated and stabilized by thin layers of serum electrolyte and proteins, and a palisade of charged polymer strands of the eGC. The ESL seems to be a respiratory organ contiguous with the flowing blood, an extension of, and a ‘lung’ in miniature. This interpretation may have far-reaching consequences for physiology.


Introduction and background: the enigmatic endothelial surface layer (ESL)
Although we understand a lot about molecular events inside cells, particularly about nuclear DNA, our grasp of phenomena occurring at the surface of cells and of collections of cells is much less sure. In particular, we have only recently begun to understand how the surface of alveoli in the lungs really operates in order to facilitate breathing (Larsson et al., 1999). But how the surface of blood vessel linings maintains necessary rheological characteristics for blood flow has largely remained a mystery. The inside or 'lumen' of blood vessels is lined first and foremost by endothelial cells which face the flowing blood. Although the endothelial cells themselves are fairly well understood as biological entities, virtually all of the loosely-adherent non-cellular layers inside of that endothelial lining are very poorly understood, particularly in terms of their physical chemistry. Blood vessels are lined by a very unusual entity which was assumed to be part solid and part liquid, which acts as a cohesive unit, and is known as the endothelial surface layer or ESL (Pries et al., 2000) (see Fig. 1).
In venous microvessels, where most studies have focused, the ESL is approximately 0.5 µm in thickness. Of that, only 50 nm is demonstrably comprised of organic molecules: a predominately polysaccharide mixture of heparan and chondroitin sulfates extending out from the endothelial membrane surface, with hyaluronic acid (HA) loosely associated with, and interior to, those polymeric sulfates. That whole region of relatively compact organic molecules is known as endothelial glycocalyx (eGC). However, the rest of the ESL, a relatively vast 450 nm in width, is 10 times larger: it is the empty region shown as a question mark in Fig. 1, 'anchored' if at all by a vanishingly dilute organic fringe of HA polymers and possibly adsorbed serum proteins . In fact, it is so dilute that, if the ESL were truly comprised predominantly of liquid, as is currently presumed, it would contain only a fraction of 0.0007 organic matter! (Secomb et al., 1998). But this is quantitatively absurd, and fails to explain how it is that the exceptionally dilute outer zone of the ESL of venous microvessels has the capacity to exclude red blood cells (RBCs), and to rebound to its original dimensions after passage of large white blood cells (Han et al., 2006) as if it were a coherent entity. Vascular biologists have attempted to explain these and other anomalies of the ESL by invoking outmoded physical chemical concepts, mainly electrostatic charge operating in high ionic strength biological fluids such as blood (Damiano and Stace, 2002;Curry and Michel, 2019).

The changing face of physical chemistry
The early founders of the cell theory of biology and the physiologists took it as axiomatic that advances in their disciplines had to draw on the enabling fields of the physical sciences. In principle no one would doubt it. But theories of physical chemistry and colloid science in aqueous media are now known to have serious flaws that have been dealt with extensively elsewhere (Hyde et al., 1996;Ninham and Nostro, 2010;Ninham, 2017;Ninham et al., 2017aNinham et al., , 2017bNinham et al., , 2017c. The defects in theory are sins of commission, and sins of omission. These developments in our understanding of forces have turned the discipline on its head. However, it turns out that an even more serious omission, the major missing X-factor, the hidden variable, lies in the effects of dissolved gas. This is something totally absent from theory. Recent developments that take this into account do allow new insights into various biological enigmas, including that of the ESL.
The resolution of the problem of pulmonary action discussed below is illustrative. Indeed, the explanation of pulmonary surfactant function and of the alveolar surface layer structure is a backdrop for untangling the ESL problem.
Pulmonary 'surfactant': new understanding For a century (Pattle, 1958), respiratory physiologists clung to the notion that pulmonary surfactants 'work' by forming a monolayer and/or a multi-bilayer lipid structure called tubular myelin at the air-fluid interface above the alveolar epithelium. However, this 'surface tension' model was shown to be physically impossible (Bangham, 1992). Therefore, the question how lung surfactants at the surface of alveoli take in O 2 and N 2 , followed by a re-opening after exhalation of CO 2 and H 2 O remained unanswered. Cryo-transmission electron microscopy (TEM) techniques revealed the real structure and mechanism of function of the surface lining of the lung (Larsson et al., 1999;Scarpelli, 2003;Follows et al., 2007;Pérez-Gil, 2008;Larsson and Larsson, 2014;Olmeda et al., 2015). The alveolar surface is comprised of 90% by the membrane-forming lipid phosphatidyl choline and several specialized surfactant proteins called A-D.
The self-assembled structure that does the trick is a prime example of non-Euclidean geometries (Hyde, 1997). Cryo-TEM on freshly opened live rabbit lungs, in 1999 (Larsson et al., 1999), revealed pulmonary surfactant to be a complex three-dimensional structure. 'A single lipid bilayer is curved in space, forming a tetragonal structure (CLP) with tubular units, the walls of which are close to planar and parallel to two orthogonal directions' (Larsson et al., 1999;Larsson and Larsson, 2014).
The proteins play an integral part, forming hexamers at the corners of the cubes (with priority over the toy maker Leggo) (see Fig. 2).
This structure opens and closes throughout the lifetime of an individual with virtually no energy cost. It opens to allow influx of O 2 and N 2 , followed by refolding accompanied by expulsion of CO 2 and water. Since then, substantially more work has been done (Scarpelli, 2003;Follows et al., 2007;Pérez-Gil, 2008;Larsson and Larsson, 2014;Olmeda et al., 2015). Experimental visualization and theory agree. It is not inconsistent with a view that the alveolar surface network (ASN) consists of a foam of nanobubbles (Pattle, 1958). The ASN is not just the liquid circulating around the nanobubbles in the alveolar foam but includes the bubbles necessarily. (We note that the nanobubbles are partially stabilized even with lung surfactant deficiencies in premature babies, reflecting the bubble-bubble fusion inhibition phenomenon [salt concentration >0.15 M]. See below) The structure of the ASN-lung surfactant region is a helpful background for understanding the ESL.

Elements of a new model of the endothelial surface layer (ESL)
Atmospheric gas: a missing player A factor completely missed in theory is a physical (as opposed to a biochemical) role for dissolved gas (Mazumdar, 2002;Ninham and Nostro, 2010;Ninham, 2017;Ninham et al., 2017aNinham et al., , 2017b). This hidden variable shows up in several ways: (1) Molecular gas (O 2 , N 2 , CO 2 ) affects long range 'hydrophobic' forces. When a solution is degassed the forces disappear. They switch off (Ninham and Nostro, 2010;Ninham, 2017;Ninham et al., 2017c). (2) Gas in water or physiological fluid exists not only in molecular form; it can be associated to form nanobubbles (Bunkin et al., 2011;Yurchenko et al., 2016). (3) Gas bubbles (and nanobubbles) in salt water will not coalesce above a salt concentration of 0.175 molar. This is exactly the ionic strength of the blood (Craig et al., 1993a(Craig et al., , 1993bHenry et al., 2007) (see Fig. 3). This still unexplained phenomenon is central to our understanding of the ESL. (4) Conceptualization and theories of self-assembled structures of lipids, surfactants, oil and electrolyte/water have usually been limited to simple Euclidean geometries: spheres, cylinders and planes. They correspond to micelles, hexagonal phases, vesicles and membranes. But the preferred states of Nature are more often non-Euclidean, bi-continuous structures that allow transport (e.g. cubosomes), as in mitochondria, or chloroplasts, or cubic (mesh) phases in conduction of the nervous impulse, and states of supra self-assembly that can transit from one form to another with extravagant ease (Hyde et al., 1996;Ninham et al., 2017aNinham et al., , 2017b. The intuition in biology comes from classical theory from which these concepts are absent.

Bubble-bubble fusion inhibition
Blood concentration of NaCl salt is around 0.15 molar, same as that in the Permian ocean from which life emerged (while the effective ionic strength, a more complex entity, is 0.175 M). There are reasons for this. Consider this experiment: bubbles emerge from a frit at the base of a column of water. They collide, fuse and grow bigger as they ascend. The column stays clear. As salt is added, and when the salt concentration reaches 0.175 molar (over a very narrow range) the bubbles no longer fuse. The column becomes opaque and densely packed with small bubbles (Craig et al., 1993a(Craig et al., , 1993bHenry et al., 2007) (see Fig. 3). The phenomenon occurs for a whole range of 1:1 electrolytes at the same concentration. It occurs for multivalent electrolytes at a different concentration, but at the identical ionic strength. In contrast, for a whole range of different salts, e.g. NaAc, it does not occur at all. There are a set of rules that determine which ion pairs 'work,' and which do not, and for mixtures (Henry et al., 2007). There are no exceptions. Nobody has any idea why. But the phenomena are indisputable.
Similar phenomena with bubble interactions occur with sugars, but at higher concentrations, typically around and greater than 1 molar (Craig et al., 1993a(Craig et al., , 1993b. We can assume that it occurs also with nanobubbles, with sizes varying from several to tens of nanometers. Nanobubbles exist and are stable against fusion at the high ionic strength of blood (Bunkin et al., 2011;Yurchenko et al., 2016). Such bubbles are further obviously stabilized by surface active solutes, sugars and serum proteins and by surfactants such as lipids.

Strange new forces: polyelectrolytes
Another player missing from theory is the very long-ranged forces of attraction between, and peculiar to, parallel conducting polymers (Richmond et al., 1972;Davies et al., 1973). These forces  exist equally for charged (linear) polyelectrolytes of the eGC and DNA (Dekker, 2001;Jimenez-Monroy et al., 2017). They are the analogs of van der Waals forces between atoms or colloidal particles.
The sources of electric current fluctuation correlations that produce the forces are the sheaf of counterions in the electrical double layer that surrounds the charged polymer core. These are very long-ranged, many-body forces. The potential of interaction per unit length is roughly ∼[1/r(ln r) 3/2 ] where r is the distance between them. They are strictly non-additive and act in concert. Accompanying this strong ordering agency are repulsive electrostatic forces between the polymers. They are much stronger when operating across a vacuum or gas rather than an aqueous medium (as usually assumed in living systems). These forces show up in unrecognized forms. Where they occur they are hidden in terms like 'anomalous water,' a 'fourth phase' of water (Pollack, 2013), and polywater.
The previously inexplicable 'exclusion zone' of the fuel cell polymer, nafion, has recently been explained by these forces (Bunkin et al., 2018). The exclusion zone repels colloidal particles, much as does our ESL. They seem to act in jellyfish to hold them together and in latex polymer suspensions. And probably they are exploited by Nature in spindle structures and ordering in the cell nucleus.
A fortiori, these forces will occur with the highly charged linear polysaccharide polymers of the eGC, most of which are sulfated, including chondroitin sulfate and heparan sulfate. They can explain how polysaccharide strands of the eGC are able to stand upright, perpendicular to the endothelial cell membrane, and parallel to one another in one (open) configuration; and lie down flat as a (closed) coating in another. If separated by gas nanobubbles, not a condensed water medium, the forces are much stronger due to the lack of screening by ions and charged molecules in solution.
Nanobubble model for the endothelial surface layer: a bicontinuous web of polysaccharide strands, water, salt and bubbles held together in a stable foam

First events: eGC-ESL nanobubble interplay
We postulate that first, CO 2 , produced as metabolic waste from endothelial cells, diffuses through the microporous 'frit' that constitutes the eGC polymer layer. In so doing, it nucleates nanobubbles of CO 2 . The stability of nanobubbles of O 2 , CO 2 and N 2 under physiological conditions is assured by the high ionic strength of biological fluids. In the nucleation process, conducting strands of the GC polymer matrix will be teased out, and form a template for the building of the ESL, and a perpendicular scaffolding for the CO 2 nanobubbles (see Fig. 4).
Such linear polyelectrolytes are subject to strong attractive cooperative long-ranged forces that align them. The opposing repulsive electrostatic forces acting across the nanobubbles are also strong (due to absence of ions in gas, as opposed to blood, which would tend to neutralize any such repulsive forces in solution). The balance of these long-ranged forces will be flexible to shear, but stiff against compression, a highly conducting electrical matrix of nanobubbles. In the surface parallel alignment of the eGC, the polymer-polymer repulsive electrostatic interactions are screened by the physiological electrolyte with a short range decay length of 0.8 nm, and coalesce due to the attractive fluctuation forces.

Hypothesis: the ESL structure
With the analogy provided by what we know of lung surfactant structure, we might reasonably postulate that the ESL is comprised predominately of CO 2 and O 2 and O 2 /N 2 nanobubbles (with H 2 O vapor) in a stable foam. It is a close-packed foam with a very thin bi-continuous layer of aqueous electrolyte and eGC strands separating the CO 2 nanobubbles, which is easy to shear but hard to compress. An essential point is that the nanobubbles in the foam will be prevented from fusion and collapse because the aqueous solution separating them is at or above physiological concentration of ∼ 0.15 M salt, and bubbles in an electrolyte concentration above 0.175 M do not fuse. Any shortfall in electrolyte concentration in vivo is taken up by the miscellany of accompanying highly charged proteins, High density lipoprotein (HDL)s and Low density lipoproteins (LDL), and act as multiply charged cations to change effective Debye leength and concentration (Mitchell and Ninham, 1978;Kékicheff and Ninham, 1990;Nylander et al., 1994). In arteries, O 2 /N 2 nanobubbles are likely predominant, while in veins there is a source of CO 2 that means CO 2 nanobubbles dominate. We imagine there is always a mix of both CO 2 and O 2 /N 2 nanobubbles. As for nitrogen/oxygen gas nanobubbles, they have an as yet less certain role (see Fig. 5).
In terms of the nature of the interface between the serum and the ESL, we characterize the ESL as a periodic steady state structure. Roughly speaking, we postulate that the number of newly-formed CO 2 nanobubbles diffusing out of endothelial cells is equivalent to the number of such nanobubbles sloughing off the ESL into the flowing blood. We can imagine that the CO 2 nanobubbles at the foam surface keep popping out, in continuous production from CO 2 gas produced inside the endothelial cells and emerging through the eGC 'frit.' Its polymers with a high proportion of sulfate groups are probably impervious to the reactive bicarbonate ion associated with high potassium ion concentration inside. The high external sodium content would act as an energetic solution 'draw' to take the bicarbonate and then CO 2 through gas. In that case, rather than an ion pump, we have a 'gas pump' (see Fig. 6).

The ESL nanobubble model explains paradoxes and anomalies
With a model structure for the ESL now constructed we proceed to challenge it. Brandon P. Reines and Barry W. Ninham The 'arginine paradox' of nitric oxide (NO) production From a classical biochemical perspective, nitric oxide (NO) is produced enzymatically from the amino acid arginine acting as a substrate for nitric oxide synthase (NOS) enzymes located in or on endothelial cells. However, most measurements in vitro and in vivo contradict the classical view: the production and biological activity of NO is not determined by cellular arginine at all, but by the amount of extracellular arginine (Vukosavljevic et al., 2006). The 'arginine paradox' is the fact that despite intracellular physiological concentration of arginine being several hundred micromoles per liter, far exceeding the ∼5 µM K M of eNOS, the acute provision of exogenous arginine still increases NO production. While a variety of explanations have been put forward to explain the arginine paradox, none has stood the test of time (Shin et al., 2011;Elms et al., 2013). However, as hydrophobic cavitation and nanobubbles have been shown to have catalytic activity (Nagase et al., 1997;Kim et al., 2001;Feng et al 2019), the fact that extracellular arginine is the main determinant of NO production might be simply explained by our nanobubble model of the ESL. Nanobubbles are an abundant source of oxidants and reductants which may potentially catalyze a variety of assumedly enzymedriven biochemical reactions (Liu et al., 2016). The arginine paradox is but one example among many where traditional biochemical thinking about enzyme action comes up short in explaining experimental data. We plan a subsequent essay exploring a new physical chemical view of catalysis.
One question which may arise is: Why would an important signaling molecule like NO, which is apparently capable of powerful effects such as vasodilation, be generated extracellularly and its production left unregulatedat the whim of nanobubblemediated catalysis? We believe the answer is provided by the recent work by Stamler's group revealing the role played by RBCs in mediating effects which had been thought due to solely to NO, but which are in reality determined by S-nitrosothiols (SNOs) (Diesen et al., 2008), Stamler has discovered that RBCs take in NO and produce SNOs, which are effectively secreted by Quarterly Reviews of Biophysics the RBCs, and actually induce vasodilationnot NO (Haldar and Stamler, 2013). The coupling of a largely random production of NO with tightly regulated processes inside of RBCs may provide a system which is carefully tailored to local oxygen demands of tissues.
Blood flow, the eGC and nitric oxide production Han et al. write that: 'A problem that has attracted widespread attention is the role of the ESL in transmitting fluid shear stress due to the flowing blood to the intracellular cytoskeleton of the endothelial cell. This problem raises a paradox since it is generally agreed that the flow within the ESL is negligible and the shear stress at the level of the cell membrane vanishingly small…' (Han et al., 2006).
In a related statement, Thi et al. highlight: 'A puzzling and still not understood consequence of eGC (endothelial glycocalyx) degradation was the observation that shear-induced NO production was greatly inhibited without apparent effect on shear-dependent vasodilation due to prostaglandin I2 release' (Thi et al., 2004).
That blood flow must transmit a mechanical signal to the actin cytoskeleton of the endothelial cell derives from the observation that flow does enhance NO release. This tends to dilate vessels and maintain normal blood pressure. These anomalies include participation of the eGC polysaccharides in NO production, but not for prostaglandin I2-mediated vasodilation. They can be explained as follows: we postulate that molecular NO is produced in most part extracellularly.
The hypothesis also explains a third important anomaly: i.e. that NO release does not correlate with actual occupancy of NOS by arginine. Instead, we can reasonably assume that most NO is produced from O 2 in O 2 /N 2 nanobubbles. These are known to provide oxidants and reductants which might interact with arginine extracellularly to produce NO (Hickok et al., 2013;Liu et al., 2016;Ahmed et al., 2018). More blood flow would tend to bring in more O 2 nanobubbles, and hence explain the correlation between increased flow, NO and vasodilation.

Soft polysaccharide strands of endothelial glycocalyx stiffen up
Although the eGC is often termed 'gel-like,' most observations show that it is surprisingly soft, more like a bed of sea grass (McLane et al., 2013;van Oosten and Janmey, 2013). Such metaphors, although impressionistic, are sometimes helpful for understanding the nature of the eGC (see, e.g. Fig. 7).
Suggestively, it sometimes has been seen to show a more refined structure, like bundles of rice plants in a paddy field in regular arrays (Squire et al., 2001). How such strands are able to stand up at all, while the main body of the eGC consists of multiple layers parallel to the cell surface was an enigma of the eGC/ ESL system. The explanation of this curious phenomenon has been given in the section 'Elements of a new model of the endothelial surface layer (ESL).' It depends on our long range attractive forces. The same forces have been shown recently to be the source of a similarly anomalous 'exclusion zone' in nafion, a fuel cell polymer which is also a sulfonated polymer (Bunkin et al., 2018).

Brief digression and technical comment: similarities with nafion
Major eGC polysaccharide polymeric components are heparan sulfate [C 12 H 19 NO 20 S 3 ] n , chondroitin sulfate [C 14 H 21 N 15 S] n and HA [C 14 H 21 NO 11 ] n , with HA as the only non-sulfated and non-cell-bound polymer. The first two have high negatively charged sulfate groups. The effective negative charge of these polyelectrolytes is reduced by binding of sodium ions (80-90%) for electrostatic interactions between the polymers in solution: the strands do not repel each other significantly in a medium with electrolytes. They do have strong short ranged repulsive hydration forces between them. This is one reason the eGC is soft and a good lubricant. The other reason is that HA is also charged with carboxylate residues, and a spacer for the hydrocarbon moieties of heparan sulfate (HS) and chondroiton sulfate (CS).
These polymers share characteristics with the hydrogen fuel cell polymer nafion, made up of multiple tetrafluoroethylenes, rather than sialic acid moieties with terminal SO 3 H sulfonic groups, which is to some extent a model for ESL barrier properties (Bunkin et al., 2018). The fuel cell properties of nafion may be shared by the eGC, especially electrical conductance.
How the ESL can be exceptionally dilute yet act as a coherent structure A confusing number cited in the literature is that the ESL above the eGC contains fractional organic matter of 0.0007, i.e. it is immeasurably dilute (Secomb et al., 1998;Pries et al., 2000). It is impossible to conceive of how a surface layer that is 99.9993% waterand/or spacecan exclude RBCs and bounce back to original volume in about half a second after passage of relatively large leukocytes (Han et al., 2006).
The matter is straightforwardly resolved if the ESL is actually a close-packed foam with a very thin bi-continuous layer of aqueous electrolyte and eGC strands separating the CO 2 nanobubbles (see Figs 5 and 6). The aqueous portion of such a close-packed foam can be as low as 0.05% of the whole ESL volume. Then the calculated mean fraction of organic matter in the thin film of water surrounding the foam bubbles drops to a more reasonable ca. 0.14%. Similar artifacts, e.g. on shear stress, and compressibility disappear with the nanobubble model.
The problem refers back to the famous work of Plateau two centuries ago.
For evolution of the transition from spherical bubbles or (oil drops in low external phase emulsions) to close-packed polyhedra at the top of Figs 5 and 6, shown further in Figs 8 and 9 (Lissant, 1966).

'High' versus 'low' salt effects on the ESL
We address here another very peculiar and important phenomenon regarding the effects of salt on the ESL.

Quarterly Reviews of Biophysics
The ESL changes quite dramatically in thickness and rigidity (Oberleithner et al., 2015) when confronted with a change in salt from 'low' to 'high' sodium plasma content: 'low' is 0.133 molar sodium, 'high' is > 0.14 molar. At first sight this is impossible. Electrostatic interactions between ions or ions and surfaces or between surfaces (double layer forces of colloid science) decay with distance L exponentially ∼exp(−KL) where K is the inverse of the Debye screening distance. For physiological saline this is about 0.8 nma couple of water molecules. It cannot be of significance. But it is! And here is how. The observation is that increase of Na + concentration from 0.133 to 0.14 M had no effect on RBC-endothelium adhesion. Above 0.14 M sudden clear-cut increased adhesion occurred (Oberleithner et al., 2015). Why?
The observed correlation between salt concentration and change in adhesion coincides with the change in bubble-bubble interactions from all fusion to no fusion. For univalent salts the transition is pretty much complete by 0.175 M, cf. Fig. 2. There, per cent bubble-bubble fusion, essentially a step function as for the red cell adhesion experiments is plotted against the concentration of sodium chloride. This (0.175 M) is different from that (0.13 to >0.14 M) of the Oberleithner et al. (2015) adhesion experiments. The discrepancy can be reconciled if we recall that for mixtures of salts the transition occurs at the same Debye length. If serum proteins and other dilute multivalent ions in the plasma are taken account of in calculating the Debye length, the range of Na + from 0.13 to >0.14 M does span the effective 0.175 M critical strength (Mitchell and Ninham, 1978;Krieg et al., 2014).
The reason for increased adhesion with increased Na + is then comprehensible. The ESL foam at the lower (0.13 M Na + ) concentration is a polydisperse and nebulous mixture, sloughing off bubbles. Nothing to adhere to there. Above the critical level (0.14 M Na + ) the ESL nanobubbles are monodisperse, close packed, and provide a firm defined base for adhesion.
The same critical bubble phenomenon with increased Na + explains other anomalies in Oberleithner's results (Oberleithner et al., 2015). One is 'an apparent discrepancy between the heparinase-induced eGC damage (stiffness decrease and height decrease) and eGC damage caused by sodium overload (stiffness increase and height decrease) …' (Oberleithner et al., 2007;Oberleithner et al., 2011;Oberleithner et al., 2015). Removal of the perpendicular templating strands of HS and CS in the eGC takes away the stabilizing framework forces; stiffness decreases and allows more rapid nanobubble escape. Sodium overload enhances bubble mono-dispersity; stiffness increases, and height decreases due to better bubble packing.
ESL phase state, vasodilation, and vascular inflammation: importance of low molecular weight thiols in opening the eGC/ESL Globally speaking, despite their apparent dissimilarity in biochemical and cellular events, we believe that vasodilation and vascular inflammation are actually closely linked in their physical chemistry. Although molecular entities that open blood vessels and those that induce inflammation are often considered vastly different in their biochemical and cellular effects, it is increasingly apparent that one and the same molecule can have both effects depending on their concentration in blood (e.g. formyl-methionine-leucyl-phenylalanine or fMLP).
In light of that, we believe that the key event in both vasodilation and vascular inflammation with increased permeability to leukocytes is 'opening of the ESL.' We contend that this is very likely due to a phase change in the entire eGC/ESLfrom a closed lamellar to open cubic or hexagonal 'cubosome' phase. It is difficult to depict this open structure but we would postulate that it is quite close to the quasi-periodic one found in electromicrographs of the eGC/ESL (Squire et al., 2001), with pore sizes of 20-40 nm. This helps explain why it is that, depending on their concentrations in vivo, many vasoactive molecules including NO can be beneficial in opening the ESL and the vessel itself, or can induce widespread damage to the vascular wall, when present in unusually high concentrations (Dorward et al., 2015).
Indeed, many vasoactive molecules share an unrecognized structural motif: their formulae contain at least one sulfurcontaining moiety. They include N-formylmethionine-leucylphenylalanine (fMLP), homocysteine, sulfatides (Li et al., 2015), virtually all of the important signaling molecules identified by Stamler (Pawloski et al., 2005;Haldar and Stamler, 2013), and possibly sulfated LDL or multiply thiolated LDL: LDL is special among lipoproteins in binding virtually any sulfur-containing molecule (Nishida and Cogan, 1970;Kim and Nishida, 1977;Kim and Nishida, 1979;Olsson et al., 1997;Camejo et al., 1998;Lundstam et al., 1999). Our model includes the implication that sulfation of polysaccharides in the eGC is critical for maintaining hydration that aligns the strands and prevents them from sticking together. The now-documented sulfur binding capacity of Apolipoprotein B of LDL becomes a key observation explaining how LDL breaches the eGC/ESL barrier, engages its receptor, and, along with facilitated entry of leukocytes, builds atherosclerotic plaques (Kim and Nishida, 1979;Olsson et al., 1997).
Mechanistically, we suspect that the reason so many vasoactive molecules are low molecular weight thiols and may also be known as atherosclerosis risk factors and/or shown to disrupt the eGC/ ESL is that they 'compete with' the hydrated sulfate-Na + -sulfate 'bridges' aligning eGC polysaccharide strands teased out from the endothelial cell surface (by CO 2 ). They can do this by inserting their own hydrated sulfur-Na + -sulfur in place of one holding the strands together. Such low molecular weight thiols would effectively pry the eGC strands apart.
A forced change in packing caused by low molecular weight thiols like fMLPs changes the anisotropic dielectric properties of the conducting eGC layers. This has a large effect on van der Waals forces between approaching neutrophils, for example. The forces can change from repulsive to attractive depending   (Haldar and Stamler, 2013).
Their chemical formulae are remarkably similar: fMLP C 21 H 31 N 3 O 5 S; Viagra C 22 H 30 N 6 O 4 S; and Levita C 23 H 32 N 6 O 4 S.
All three are very similar with much the same molecular weight (437 for fMLP) to typical lipids. The sulfate moiety facilitates adsorption in the foam of the ESL and of the eGC. There they induce a transition to a bicontinuous open cubosomal phase that allows uptake of N 2 /O 2 nanobubbles and production of nitric oxide.
A further consequence is that these molecules, in their surfactant guise, can in succession be further taken up into cell lipid membranes. There they induce transitions to mesh phases involved in transmission of the nervous impulse, the desired response.
Taurine C 2 H 7 NO 3 S (MW 125) probably dimerizes and have the same physical effects.
Cialis, a precursor erectile drug, C 22 H 19 N 3 O 4 and candesartan, a popular drug for heart disease C 24 H 20 N 6 O 3 will affect the same physical changes and react with sulfate moieties of the eGC polymers.

Low molecular weight thiols induce a phase change in the eGC/ESL
The usual focus on specific formulae and binding sites of such and related molecules masks a more general overarching phenomenon. What is occurring is a phase change triggered by the low molecular weight thiols such as fMLPs in the microstructure of the eGC. This is a re-run of the phase changes in self assembled cell lipid membrane structures that underlies anesthetic action, propagation of the nerve impulse, immuno-suppression by cationic surfactants and pheromone action (Ninham et al., 2017a(Ninham et al., , 2017b. Transitions from closed e.g. multiple lamellae, to open bicontinuous phases occur with extravagant ease, induced by cosurfactants or changes in physico-chemical conditions (Ninham et al., 2017a(Ninham et al., , 2017b. There is a unifying predictive theory and these matters are well understood. There is a disconnect in thinking about lipid/surfactant selfassembly versus that of polyelectrolyte such as polysaccharide phase behavior (as for the eGC). Yet local curvature versus global packing constraints set structure for polyelectrolytes like eGC polysaccharides is governed by the same constraints as for surfactants and lipids. Corresponding to closed lamellae versus open cubic phases in lipid/surfactant systems we have closed polymer lamellae-like versus open cubosomes in polyelectrolyte systems. The cubosome phases of polymers are very rigid as compared with lamellar states of polymers, besides the obvious unrecognized differences between open and closed physical states. The cubosome literature in cell biology is large and exploited in the pharmaceutical field for drug delivery. Of particular interest in the context of the ESL is the facts that pore sizes are characteristically around 20-40 nm (Squire et al., 2001), that a cubosomal structure for the ESL is very rigid compared with the lamellar state, and the transition can be induced by a wide variety of low molecular weight thiols, small peptides or surfactants. Most share with fMLP the sulfur-containing moiety for hydration compatibility with the ESL polyelectrolytes, facilitating opening of the eGC/ESL.

Discussion: implications for physiology and immunology
Respiratory and vascular physiology are usually treated as separate. The known stability of nanobubbles under physiological conditions suggests now that the two systems are linked. The notion that O 2 is transported bound to hemoglobin is a fact. The notion of a complementary delivery mechanism arises if O 2 (and N 2 ) nanobubbles exist and are stable in physiological medium. A related, open question is how CO 2 , a byproduct of metabolism, exits cells into the circulatory system and on to the lungs for expulsion. It is known that CO 2 diffuses into RBCs, where it is converted to carbonic acid by the enzyme carbonic anhydrase and then converts to HCO 3 (Krieg et al., 2014). But this may not be the only escape route for CO 2 . A foam of CO 2 bubbles that form the ESL is another. Such a model resists compression, but is simultaneously susceptible to shear and sloughing off of nanobubbles into blood plasma flow.
At the same time, an optimal level of vessel wall flexibility would be preserved, but not so rigid as to create problems such as limitations on O 2 delivery to tissues and stiffening of vessel walls. The three qualitatively new factors that explain the ESL are: the existence of stable nanobubbles at physiological ionic strength, the phenomenon of bubble-bubble fusion inhibition above 0.175 M, and long range fluctuation forces between polyelectrolytes (Richmond et al., 1972;Davies et al., 1973). The latter, known almost since the discovery of van der Waals forces seems to have fallen through the cracks and been ignored.
Some questions on respiratory and vascular physiology and on immunology can now be revisited. Nature seems to have taken care to put long stretches of positively charged amino acids into hormone-like signaling molecules, like chemokines and cytokines. The function of their cationicity (and specific hydration) was attributed to the requirement that such molecules interact with negatively charged regions of the cell surface, particularly its sulfate-rich glycocalyx. That is a tautology. But an explicit reason associated with nanobubbles can be adduced. The cationic polymer-anionic heparin sulfate complex residues in the eGC would collapse the ESL (Manchanda et al., 2018). The repulsive electrostatic interactions due to the heparin across the (electrolyte-free) nanobubbles that, with the opposing attractive ionic fluctuation forces that stabilize them, are switched off. Therefore, the positive charge matters as long suspected, but not in the way long supposed: in the assumed aqueous system, screening of the charges occurs due to ions, but in the gas eGC/ESL system we propose, the positive charges would definitely collapse the whole system.
It seems that nanobubbles of oxygen, nitrogen and carbon dioxide play a so far previously hidden and complementary role in transport and delivery that is excluded from the conventional canon. It is conceivable that the ESL is part of a connected extension of the lungs like the lymphatic system that we have not previously recognized. The sizes of the compartments in the lungs are around 40 nm, same more or less as those seen in EM images of ESL (20-40 nm) (Reitsma et al., 2007). In this scenario, as is usual in physiology, nitrogen, 80% of atmospheric gas taken in is the odd man out. The arguments above would imply that it too forms nanobubbles, not just a molecular solution, but a mixture with oxygen and they would be a part of the action. The solubility of nitrogen in fact is five times that of oxygen, the connection to the problem of the 'bends' (Craig et al., 1993a, 1993b, Arieli, 2015Arieli, 2017).
The structure of the ESL has been a frustrating puzzle. A gallimaufry of enigmas all point to a continuously produced ordered Quarterly Reviews of Biophysics foam of nanobubbles as the answer. For the ESL the remarkable phenomenon of bubble-bubble fusion inhibition at and around physiological concentration seems an obvious key.
Strong long-ranged polyelectrolyte fluctuation forces seem also to be necessary to explain other curious phenomenajellyfish, for which the umbrella is variously estimated to be up to 99% water. An extremely dilute matrix permeated by stinging polyelectrolytes just like the ESL seems a more probable explanation. The anomalous exclusion zone at the surface of nafion, the (sulfated) fuel cell polymer, repels colloidal particles or macromolecules, just as does the ESL (Davies et al., 1973;Bunkin et al., 2018). Their shared electrochemical properties with the ESL open up promising new areas.