Cancerous Cells May Continuously Change Their Glycocalyx

Glycocalyx

Degradation of the glycocalyx layer covering the endothelium, with shedding of endothelial cells and loss of tight junctions between the cells, results in increased capillary leak and the development of tissue edema.

From: Perioperative Medicine (Second Edition) , 2022

Basic structure and function of cells

Susan Standring MBE, PhD, DSc, FKC, Hon FAS, Hon FRCS , in Gray's Anatomy , 2021

Cell coat (glycocalyx)

The external surface of a plasma membrane differs structurally from internal membranes in that it possesses an external, fuzzy, carbohydrate-rich coat, the glycocalyx, composed of the carbohydrate portions of glycoproteins and glycolipids embedded in the plasma membrane (see Fig. 1.3). The cell coat forms an integral part of the plasma membrane, projecting as a diffusely filamentous layer 2–20   nm or more from the lipoprotein surface.

The precise composition of the glycocalyx varies with cell type; many tissue and cell type-specific antigens are located in the coat, including the major histocompatibility complex of the immune system and, in the case of erythrocytes, blood group antigens. The glycocalyx therefore plays a significant role in organ transplant compatibility. The glycocalyx found on the apical microvilli of enterocytes, the cells forming the lining epithelium of the intestine, consists of enzymes involved in the digestive process. Intestinal microvilli are cylindrical projections (1–2   μm long and about 0.1 μm in diameter) forming a closely packed layer called the brush border that increases the absorptive function of enterocytes (seeFig. 1.26).

The glycocalyx plays a significant role in maintenance of the integrity of tissues and in a wide range of dynamic cellular processes, e.g. serving as a vascular permeability barrier and transducing fluid shear stress to the endothelial cell cytoskeleton (Weinbaum et al 2007). Disruption of the glycocalyx on the endothelial surface of large blood vessels precedes inflammation, a conditioning factor of atheromatosis (e.g. deposits of cholesterol in the vascular wall leading to partial or complete obstruction of the vascular lumen).

Reactive Oxygen and Nitrogen Species and Liver Ischemia-Reperfusion Injury: An Overview

Fabienne T.E. Alban , ... Michal Heger , in The Liver, 2018

Oxidative Degradation of the Endothelial Glycocalyx

The glycocalyx (GCX) is a network of proteoglycans (PGs) and glycosaminoglycans (GAGs) lining the sinusoidal endothelial cells (SECs). ²⁹ The PGs, which belong to the syndecan and glypican family, form the backbone of the GCX with branches made of GAGs. The predominant GAGs are heparan sulfate (HS) and chondroitin sulfate (CS), attached to the PGs. Hyaluronic acid (HA), another type of GAG, is not attached to the PGs and intercalates into the GCX through its interaction with endothelial HA receptors and CS chains. ¹ The GCX plays a vital role in maintaining microvascular homeostasis by preventing leukocyte and platelets adhesions and through regulation of vascular permeability and tone. ¹

The GCX is vulnerable to oxidative and nitrosative stress during hepatic ischemia/reperfusion. The notable ROS involved in this process are OH, $\text{C}{\text{O}}_3^{·-}$ , and HOCl, which are capable of causing oxidative fragmentation in HS, CS, and HA, leading to the degradation of the sinusoidal endothelial GCX. ¹ Detailed mechanisms of the detrimental effect of these reactive species have been reviewed by van Golen and coworkers. ¹ GCX degradation results directly in perturbation of microvascular homeostasis by enabling leukocyte and platelet adhesion and perturbed fluid exchange (edema) ¹ as a result of compromised vascular permeability. Additionally, an immune response is elicited by the circulating GCX fragments via activation of leukocytes and ECs, amplifying the hepatic I/R-induced injury through exacerbated ROS/RNS production. ¹ Accordingly, oxidative degradation of GCX contributes to the pathophysiology of hepatic I/R injury.

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The Cell and Its Functions

John E. Hall PhD , in Guyton and Hall Textbook of Medical Physiology , 2021

Membrane Carbohydrates—The Cell "Glycocalyx."

Membrane carbohydrates occur almost invariably in combination with proteins or lipids in the form ofglycoproteins orglycolipids. In fact, most of the integral proteins are glycoproteins, and about one-tenth of the membrane lipid molecules are glycolipids. Theglyco- portions of these molecules almost invariably protrude to the outside of the cell, dangling outward from the cell surface. Many other carbohydrate compounds, calledproteoglycans—which are mainly carbohydrates bound to small protein cores—are loosely attached to the outer surface of the cell as well. Thus, the entire outside surface of the cell often has a loose carbohydrate coat called the glycocalyx.

The carbohydrate moieties attached to the outer surface of the cell have several important functions:

1.

Many of them have a negative electrical charge, which gives most cells an overall negative surface charge that repels other negatively charged objects.

2.

The glycocalyx of some cells attaches to the glycocalyx of other cells, thus attaching cells to one another.

3.

Many of the carbohydrates act asreceptors for binding hormones, such as insulin. When bound, this combination activates attached internal proteins that in turn activate a cascade of intracellular enzymes.

4.

Some carbohydrate moieties enter into immune reactions, as discussed inChapter 35.

The Critically Ill Patient

Michael R. Pinsky , in Critical Care Nephrology (Second Edition), 2009

CELL ADHESION AND ORGAN FAILURE

A complex and highly variable surface, or glycocalyx, covers the free cell surface of most cells. This covering is composed of numerous lipid-carbohydrate-protein moieties that extend out from the cell, attach to the cell surface, and may cross the cell membrane once or several times. This surface is not a passive barrier to diffusion, although it can function in that way as well. It is primarily a major recognition and attachment site for cell interactions that, for inflammation, translates into a ligand binding site with functional inflammatory transduction characteristics. Such binding must activate an intracellular process that results in upregulation of gene expression, synthesis of new protein, and phenotypic changes characteristic of inflammation that can be measured outside the cell.

As an initiating receptor, the cell surface receptor CD14 plays a pivotal role in the recognition of many microbiological toxins, host-derived mediators, and, probably, more molecular species with immune-modulating characteristics. To activate the CD14 receptor, LPS in the bloodstream binds to an LPS binding protein (LBP), the complex of which can then bind to CD14. ²⁶ CD14 exists as a superficial receptor that has no cross-membrane component. ²⁷ Presumably, CD14 functions to present the LPS-LBP complex to Toll-like receptor-4 (TLR-4), which internalizes the LPS moiety. Cells without CD14 do not react to LPS exposure. ²⁸ Interestingly, CD14 can be shed into the circulation and bind LPS-LBP complexes. This trimer can bind to otherwise non-CD14 cells, conferring LPS sensitivity. The significance of this finding is currently unknown but is probably a cause of increased apoptosis of otherwise non–immunologically competent tissues, such as cardiac myocytes. The cell surface complex of LPS, LBP, and CD14 interacts with the transmembrane protein TLR-4 to induce intracellular signal transduction for the synthesis of new TNF and, probably, other pro-inflammatory cytokines (Table 1-2). ²⁷ The initial central point in the first steps of intracellular activation is activation of serine kinases. Activated serine kinases eventually activate nuclear factor-κB (NF-κB), which cleaves its inhibitory component, migrates into the nucleus, and binds to promoter sites on the genome coding for the synthesis of messenger RNA of various inflammatory protein species, including TNF.

Release of pro-inflammatory cytokine stimulates endothelial cells and circulating immune effector cells to change the expression of their cell surface adhesion molecules. The initial proadhesion cell surface receptors expressed are from the family of molecules called selectins. They allow for loose binding of circulating PMNs to the endothelium. The initial site of binding is in the postcapillary venule, where radial dispersive forces and low flow rate–shear forces promote cell-cell contact. This initial binding is weak, and if local endothelial activation induces expression of L-selectin–specific ligands along the endothelial surface, normal vessel flow induces the PMNs to roll along the vascular endothelium. If there is minimal endothelial activation, the PMNs dislodge and reenter the circulation. This transient binding of PMNs to the endothelium is the cause of initial leukopenia in response to dialysis-induced PMN activation and other related phenomena. If the endothelium has been strongly stimulated either by systemic pro-inflammatory mediators or local injury/inflammation, it will express a second family of cell adhesion molecules called integrins, of which intercellular adhesion molecules (ICAMs) are a subclass. ICAM expression on vascular endothelial cells occurs at the expense of selectin expression. Other integrins, such as CD11b and CD18, are upregulated on PMNs during sepsis. The strongly negatively charged selectins are cleaved into the circulation early in a septic challenge and may represent a major component in the initial acidosis seen in sepsis. ²⁸

Endothelial cell activation induces expression of L-selectin and, if sustained, of ICAM. Both adhesion molecules promote platelet binding rather than repulsion, which is the reason for the development of the procoagulant activity of the endothelium during inflammation. Activated endothelium expresses IL-8, a potent PMN chemoattractant, into its microenvironment. IL-8 suppresses the subsequent activation of immune effector cells in response to subsequent exposure to TNF. ICAM expression promotes firm binding of those PMNs that are also activated. Tight adhesion mediated through ICAM binding allows for prolonged PMN-endothelial contact. Activated PMNs bound to activating endothelium is a deadly combination for the endothelial cell. Release of toxic oxygen species and proteases by the PMNs into the microenvironment between these two cell types promotes lipid peroxidation and damage to the endothelial barrier of the vessel. In a less well-defined process, chemoattractants within the site of inflammation induce the migration of other PMNs into the parenchyma once endothelial damage has occurred.

Using the Goris Organ Failure Score in septic patients with end-stage liver failure, Rosenbloom and associates ¹⁰ correlated change in adhesion molecule activity with subsequent development of organ failure. ⁸ They found that mild sepsis was associated with slight elevations in L-selectin on PMNs, whereas severe sepsis was associated with a decreased L-selectin expression, a markedly increased CD11b expression, and a slightly increased CD35 expression (complement-binding receptor). Furthermore, although both IL-6 serum levels and CD11b PMN levels were highly variable among patients and even variable over time, most of the variability in CD11b expression could be explained by paired variations in IL-6. Finally, levels of CD11b and CD35 on circulating PMNs predicted subsequent organ failure. Accordingly, variations in CD11b over time related to variations in IL-6 serum level and to the severity of intravascular inflammation, as assayed by CD11b upregulation predicted subsequent MODS.

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Fluid Distribution in the Fetusand Neonate

Richard A. Polin MD , in Fetal and Neonatal Physiology , 2022

Endothelial Glycocalyx Layer

The endothelial glycocalyx layer (EGL) is well-known intravascular structure in adults that is also found in the human placenta ⁶¹ and umbilical vein. ⁶² Movement of fluid, including proteins, between the intravascular and the ISF compartments is crucially dependent on the capillary endothelium and overlying capillary endothelial glycocalyx, which form the EGL. ⁶³ The EGL is a major determinant of vascular permeability and plays a crucial role in movement of fluid between the intravascular and ISF compartments, which results in normal fluid homeostasis. The endothelial glycocalyx consists of glycoproteins and proteoglycans containing glycosaminoglycans attached to the endoluminal surface of the capillary endothelium. Albumin is contained within the glycocalyx layer, and the EGL requires a normal level of plasma albumin to function. The vascular endothelium/glycocalyx barrier is freely permeable to water, semipermeable to albumin, but impermeable to large protein molecules (>70 kDa) in plasma.

The hydrostatic and oncotic pressure gradients between the lumen of the blood vessel and the interstitial space depend largely on the endothelial glycocalyx. The oncotic pressure difference is not built up between the intravascular and the interstitial tissue spaces, but within a small protein-free zone beneath the glycocalyx surface layer (subglycocalyx space). The oncotic pressure difference is built up between the adsorbed albumin in the soluble EGL and a small protein-free zone (subglycocalyx space) leading to the revised Starling's equation. ⁶⁴

The traditional Starling equation follows:J _v =K _f ([P _c −P _i] −σ[π_c − π_i]), whereJ _v is the net fluid movement between compartments and ([P _c −P _i] −σ[π_c − π_i]) is the net driving force,P _c is the capillary hydrostatic pressure,P _i is the interstitial hydrostatic pressure, π_c is the capillary oncotic pressure, π_i is the interstitial oncotic pressure,K _f is the filtration coefficient—a proportionality constant—andσ is the reflection coefficient. Starling stated that these forces are balanced. Based on EGL, the revised Starling's equation incorporates π _g (glycocalyx oncoticpressure) instead of π_i, and is stated asJ _v =K _f ([P _c −P _i] −σ[π_p − π _g ])(Fig. 105.7). ⁶⁴

Volume 1

Wilhelm Kriz , Brigitte Kaissling , in Seldin and Giebisch's The Kidney (Fifth Edition), 2013

Reabsorption of Water and Solutes

The plasma membrane of the microvilli is covered by a glycocalyx containing hydrolases (phosphatases, peptidases, nucleotidases) which cleave their substrates in the tubular fluid (ecto-enzymes). The microvillous membrane holds a large variety of transport proteins for uptake of water and solutes from the tubular fluid. The density of a given transport protein in the microvillous membrane can be dissimilar along the segments of the proximal tubule and among nephron generations. Many of the transport proteins are anchored by adaptor proteins, such as PDZ-proteins and NHERF1/2, to the underlying apical scaffold. ^{372,373,378,379}

Transcellular water reabsorption in the proximal tubule is mediated by the constitutive water channel, aquaporin 1 (AQP1) located in the microvillous- and basolateral plasma membrane domains. ^380–384 Orthogonal arrays of intramembrane particles in the basolateral membranes of S3 of mice ³⁸⁵ are associated with another water channel, AQP4, AQP7, which is probably involved in the reabsorption of glycerol (see review in ³⁸⁶ ) and is expressed in the brush border, especially of S3 in rats and mice, as shown by immunocytochemistry. ^387,388

Sodium-coupled solute uptake from the lumen into the cells is mediated by co-transport proteins located in the plasma membrane of the microvilli. The proximal tubule usually recovers all filtered glucose. The sodium–glucose co-transporter SGLT2 is found primarily in S1, and is responsible for 90% of glucose reabsorption. SLGT1 is located in S3, and is responsible for only 10% of reabsorption (reviewed by Hediger and Rhoads ^389,390 ). SGLT1 is more highly expressed in females than in males. ³⁹¹ The exit of glucose across the basolateral plasma membrane occurs by the glucose transporters GLUT2 (low affinity in S1) and GLUT1(high affinity in S3). ³⁹²

Inorganic phosphate (Pi) transport is mediated by at least three different brush border Na⁺/P(i) co-transporter proteins, the electrogenic transporter NaPi IIa, Pit-2, and the electroneutral transporter NaPi Iic. ^393,394 Their expressions and activities appear to be tightly regulated.

Low dietary intake of Pi increases mRNA and brush border expression of NaPi IIa. ³⁹⁵ High dietary Pi intake, parathyroid hormone (PTH) and activation of dopamine receptors ³⁵⁸ rapidly downregulate NaPiIIa-mRNA ³⁹⁵ and NaPiIIa in the brush border, ^396–398 and induce phosphaturia. Downregulation of NaPiIIa in the brush border involves receptor-mediated endocytosis (see above) and subsequent lysosomal degradation. ^{355,356,358,399,400}

The passage of NaPi-IIa across the successive endocytotic compartments namely, the megalin-containing clefts, the clathrin-coated-vesicle compartment, ⁴⁰¹ through the early and late endosomal compartment, and finally its disposal in lysosomes, where NaPi-IIa is degraded, has been tracked by immunofluorescence. ³⁰¹ The shifting of the protein through the early endocytotic compartments goes along with a dramatic, rapidly transient expansion and remodeling of the vacuolar apparatus in the subapical compartment ³⁵⁶ (Figure 20.45). PTH also reduces Pit-2 expression and activity, whereas NaPi-IIc is inhibited and internalized with a delay of several hours after PTH application. ³⁹⁴

Recently Klotho has been recognized as a phosphatonin, and an important regulator of phosphate homeostasis. In partnership with the FGF-R, Klotho functions as an obligate co-receptor for FGF23. ⁴⁰² Secreted soluble Klotho inhibits Pi transport by altering the trafficking of the proximal tubule Na-coupled phosphate transporter. ⁴⁰²

Neutral amino acids, which represent about 80% of circulating amino acids, are transported by the low affinity Na⁺-co-transporter B(0)AT1, located in the early proximal tubule. The high affinity transporter B(0)AT3 is located in the late proximal tubule, at least in mice. In addition, there are several other apical and basolateral amino acid transporters (for a recent review see ⁴⁰³ ). Similarly the short-chain peptide, di-, and tri-peptide carriers PEPT1, high capacity, low affinity, and PEPT2 low capacity, high affinity are located in mainly S1 and S3, respectively. ^404,405

Secretion of organic amphiphilic electrolytes from the blood into the tubular fluid is a pathway for clearance and detoxification of xenobiotics and drugs, including diuretics. ^{325,406–410} The uptake into the proximal tubule epithelium proceeds via multispecific organic anion transporters (OAT) and organic cation transporters (OCT) in the basolateral membrane domain. The majority of members of the OAT- and OCT-family have been immunolocalized to the basolateral cell membrane of S3 proximal tubule, ^411–413 yet OAT 1 has been detected mainly in S2, ⁴¹⁴ a few also in S1. The expression of the OATs and OCTs is strongly regulated by sex hormones. ^415–420

The export into the tubular lumen of both conjugated and unconjugated lipophilic anionic substrates involves various OATs and primarily active transporters with ATP-binding casette motifs, belonging to the MRP-family, ⁴²¹ and located in the brush border membrane of S1, S2, and S3 proximal tubule segments. ^421,422

The role of basolateral endocytosis is interesting, since the basolateral cell membrane is the site of different hormone receptors, ³⁶⁸ e.g., the insulin receptor. After binding to the receptors peptide hormones seem to be, at least in part, taken up by the cells and are transported to the lysosomes. ³⁶⁹

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Investigation of haemostasis

Mike Laffan , Richard Manning , in Dacie and Lewis Practical Haematology (Tenth Edition), 2006

Endothelial Cell Function ¹

The luminal surface of the endothelial cell is covered by the glycocalyx, a proteoglycan coat. It contains heparan sulphate and other glycosamino-glycans, which are capable of activating antithrombin, an important inhibitor of coagulation enzymes. Tissue factor pathway inhibitor (TFPI) is present on endothelial cell surfaces bound to these heparans but also tethered to a GPI (glycophosphoinositol) anchor. The relative importance of these two TFPI pools is not known. Endothelial cells express a number of coagulation active proteins that play an important regulatory role such as thrombomodulin and the endothelial protein C (PC) receptor. Thrombin generated at the site of injury is rapidly bound to a specific product of the endothelial cell, thrombomodulin. When bound to this protein, thrombin can activate PC (which degrades factors Va and VIIIa) and a carboxypeptidase (which inhibits fibrinolysis; discussed later). Thrombin also stimulates the endothelial cell to produce plasminogen activator. The endothelium can also synthesize protein S, the cofactor for PC. Finally, endothelium produces von Willebrand factor (VWF), essential for platelet adhesion to the subendothelium. This is both stored in specific granules called Weibel Palade bodies and secreted constitutively, partly into the circulation and partly toward the subendothelium. The expression of these and other important molecules such as adhesion molecules and their receptors are modulated by inflammatory cytokines. The lipid bilayer membrane also contains ADPase (adenosine diphosphatase), an enzyme that degrades ADP (adenosine diphosphate), which is a potent platelet agonist (see p. 430). Many of the surface proteins are found localised in the specialized lipid raft invaginations called caveolae. ²

The endothelial cell participates in vasoregulation by producing and metabolising numerous vasoactive substances. On one hand it metabolises and inactivates vasoactive peptides such as bradykinin; on the other hand, it can also generate angiotensin II, a local vasoconstrictor, from circulating angiotensin I. Under appropriate stimulation the endothelial cell can produce vasodilators such as nitric oxide (NO) and prostacyclin or vasoconstrictors such as endothelin and thromboxane. These substances have their principal vasoregulatory effect via the smooth muscle but also have some effect on platelets.

The subendothelium consists of connective tissues composed of collagen (principally types I, III, and VI), elastic tissues, proteoglycans, and noncollagenous glycoproteins, including fibronectin and VWF. After vessel wall damage has occurred, these components are exposed and are then responsible for platelet adherence. This appears to be mediated by VWF binding to collagen, particularly under high shear rate, and also to the microfibrils, which have a greater affinity for VWF under some conditions. VWF then undergoes a conformational change, and platelets are captured via their surface membrane glycoprotein Ib binding to VWF. Platelet activation follows, and a conformational change in glycoprotein IIbIIIa allows further, more secure, binding to VWF via this receptor as well as to fibrinogen. At low shear rates platelet binding directly to collagen appears to dominate. ³

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Investigation of haemostasis

Michael A. Laffan , Richard Manning , in Dacie and Lewis Practical Haematology (Eleventh Edition), 2012

Endothelial Cell Function

The luminal surface of the endothelial cell ¹ is covered by the glycocalyx, a proteoglycan coat. It contains heparan sulphate and other glycosaminoglycans, which are capable of activating antithrombin, an important inhibitor of coagulation enzymes. Tissue factor pathway inhibitor (TFPI) is present on endothelial cell surfaces bound to these heparans but also tethered to a glycophosphoinositol (GPI) anchor. The relative importance of these two TFPI pools is not known. Endothelial cells express a number of coagulation active proteins that play an important regulatory role such as thrombomodulin and the endothelial protein C (PC) receptor. Thrombin generated at the site of injury is rapidly bound to a specific product of the endothelial cell, thrombomodulin. When bound to this protein, thrombin can activate PC (which degrades factors Va and VIIIa) and a carboxypeptidase which inhibits fibrinolysis (discussed later). Thrombin also stimulates the endothelial cell to produce tissue plasminogen activator (tPA). The endothelium can also synthesize protein S, the cofactor for PC. Finally, endothelium produces von Willebrand factor (VWF), which is essential for platelet adhesion to the subendothelium and stabilizes factor VIII within the circulation. VWF is both stored in specific granules called Weibel Palade bodies and secreted constitutively, partly into the circulation and partly toward the subendothelium where it binds directly to collagen and other matrix proteins. The expression of these and other important molecules such as adhesion molecules and their receptors are modulated by inflammatory cytokines. The lipid bilayer membrane also contains adenosine diphosphatase (ADPase), an enzyme that degrades adenosine diphosphate (ADP), which is a potent platelet agonist (see p. 434). Many of the surface proteins are found localized in the specialized lipid rafts and invaginations called 'caveolae', which may provide an important level of regulation. ²

The endothelial cell participates in vasoregulation by producing and metabolizing numerous vasoactive substances. On the one hand, it metabolizes and inactivates vasoactive peptides such as bradykinin; on the other hand, it can also generate angiotensin II, a local vasoconstrictor, from circulating angiotensin I. Under appropriate stimulation the endothelial cell can produce vasodilators such as nitric oxide (NO) and prostacyclin or vasoconstrictors such as endothelin and thromboxane. These substances have their principal vasoregulatory effect via the smooth muscle but also have some effect on platelets.

The subendothelium consists of connective tissues composed of collagen (principally types I, III and VI), elastic tissues, proteoglycans and non-collagenous glycoproteins, including fibronectin and VWF. After vessel wall damage has occurred, these components are exposed and are then responsible for platelet adherence. This appears to be mediated by VWF binding to collagen. VWF then undergoes a conformational change and platelets are captured via their surface membrane glycoprotein Ib binding to VWF. Platelet activation follows and a conformational change in glycoprotein IIbIIIa allows further, more secure, binding to VWF via this receptor as well as to fibrinogen. At low shear rates (<1000 s⁻¹) platelet binding directly to collagen appears to dominate. ³

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Anesthesia for Spine Surgery and the Prevention of Complications

Ehab Farag , ... Zeyd Ebrahim , in Benzel's Spine Surgery, 2-Volume Set (Fourth Edition), 2017

Perioperative Fluid Management and Glycocalyx

Perioperative fluid management is one of the key factors in maintaining the integrity of EG. It is well known that iatrogenic acute hypervolemia can lead to the release of atrial natriuretic peptide (ANP). ANP induces shedding of EG components, mainly syndecan-1, thereby increasing shifts of fluid and macromolecules into the interstitial space. Thus, the ability of ANP to increase capillary permeability to water, solutes, and macromolecules might be at least partially explained by its capacity to disturb the EG structure. The average insensible fluid loss is only about 0.5 mL/kg/h via skin and airways in the awake adult. During abdominal surgery, insensible fluid loss increased to only 1 mL/kg/h. Avoiding hypovolemia and hypervolemia, which includes a careful indication for perioperative fluid management, is an important element to maintain a healthy EG and thereby to limit perioperative fluid and protein shifts into the interstitial space. An intact ESL is essential to avoid excessive tissue edema. Therefore, a goal=directed fluid approach is essential to maintain normovolemia and ESL integrity, reducing postoperative complications like anastomotic leaks, nausea and vomiting, infections, and pulmonary complications. ^27-31

Many of these complications may result from excessive use of crystalloids. Of note, 80% of the infused crystalloids are distributed into interstitial tissues under all conditions. Therefore, the use of crystalloid at the rate of 1 to 2 mL/kg/h for maintenance and the use of iso-oncotic colloid like albumin for the replacement of blood loss may be the ideal for fluid management during spine surgery.

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Volume 1

Richard R. Drake , ... Peggi M. Angel , in Molecular Imaging (Second Edition), 2021

N-Linked Glycans

There is a dense outer layer of complex carbohydrates on the cell surface termed the glycocalyx that is broadly involved in extracellular matrix organization, molecular architecture, and immune recognition. Structurally, the glycocalyx is comprised of multiple types of oligomeric and protein-linked complex carbohydrates such as: N- and O-linked glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids, linked with a meshwork of ECM and cellular proteins. Currently, IMS analysis of tissue glycans has focused on N-glycans [14,17,91,145], the glycan structural class linked to asparagine residues on protein carriers. This is largely due to the availability of the enzyme peptide N-glycosidase F (PNGase F), which cleaves the sugars intact from the asparagine linkage, allowing direct tissue profiling of the released glycans. Changes in N-linked glycosylation are well documented to affect signal transduction, cellular mobility and invasiveness, and immune properties [14], and they are inherently linked with altered tissue microenvironments associated with disease. The majority of studies have focused on different cancer types using FFPE blocks direct from pathology and TMAs due to the availability of archival tissue blocks linked to clinical data. Thus, N-linked glycans present in regions of tumor can be compared to adjacent regions of nontumor tissue and stroma [17,88–91,146]. It can also be linked with other IMS proteomic analyses, as the glycans can be imaged, then the same tissue prepared for IMS of trypsin or collagenase derived peptides [78,147]. Even though N-glycan studies can be done using fresh frozen tissues, the size and structural properties have restricted detection by DESI or SIMS methods, and MALDI IMS is the current ionization of choice.

There is much diversity in the structure and composition of N-glycans, reflective of the nontemplate-driven biosynthesis and processing of sequential glycosidase and glycosyltransferase reactions [148]. Structural assignment of detected N-glycans is based on accurate mass, orthogonal tandem mass spectrometry verification, and the biological context of the N-glycan biosynthesis and processing pathways. Other enzymes can be used in this verification, like glycosidases targeting specific terminal sugars, and a different enzyme that cleaves N-glycans with specific N-acetylglucosamine-fucose linkages to the asparagine [149]. A chemical derivatization method to stabilize terminal sialic acids has also found utility for IMS [90]. An example N-glycan image of prostate tissues treated with the sialic acid stabilizing agent is shown in Fig. 17.5. This has addressed a long-standing limitation of standard MALDI-TOF instruments in that the heat and pressure of the ionization process can dissociate sialic acids. Certain MALDI-FTICR instruments have a cooling gas present during the ionization process,which mitigates much of this loss. The overall method workflows have been well standardized, and work well with MALDI-TOF and MALDI-FTICR instruments. Depending on the tissue and disease state, generally 40-60   N-glycoforms are routinely detected, although up to 100 or more can be detected in some cancer tissues.

Figure 17.5. Example N-glycan imaging mass spectrometry using a MALDI QTOF (timsTOF fleX, Bruker Scientific) on FFPE prostate tissues treated with amidation reactions to stabilize sialic acids. The images highlight the regiospecific localization of sialic acid isomers in tissue. For each tissue image, two stromal glycans are highlighted, a Hex5HexNAc4Fuc with a single alpha-2,3 linked sialic acid (in green) and a Hex5HexNAc4 with a single alpha-2,6 linked sialic acid (in blue). (A) An H&amp;E image of benign disease (left) and tumor (right) from the same donor. (B) A Hex7HexNAc6Fuc with a single alpha-2,6 linked sialic acid highlighted in the tumor region (in red). (C) A Hex7HexNAc6Fuc with a single alpha-2,3 linked sialic acid highlighted in the tumor region (in white/gray). (D) An overlay of the two tumor glycans, illustrating minimal overlap of the two isomers within the tumor region.

In cancer tissues, this method has been utilized to spatially localize specific glycan structures to tissue-specific regions (tumor, stroma, and necrosis). A study analyzing the glycosylation patterns of breast cancer tissues found structural glycan classes detected in the tumor and stromal regions were typically classified as high mannose or branched glycans. Additionally, glycans found in necrotic regions displayed limited branching, contained sialic acid modifications and lacked fucose modifications. While this phenomenon was initially classified in breast cancer tissues, it was also seen in cervical, thyroid, and liver cancer samples [86]. Another study also analyzing breast cancer glycosylation identified shared and divergent glycosylation patterns among HER2+ and triple negative breast cancers. MSI glycan analysis revealed a series of high-mannose glycans that were predominantly associated with tumor regions, as well as more highly branched and fucosylated glycans in higher grade tumors. In addition to this, a series of polylactoseamine glycans with increased expression in HER2+, triple negative, and metastatic breast cancers were identified [150]. Example IMS data for a triple negative breast cancer tissue with extensive regions of necrosis is shown in Fig. 17.6.

Figure 17.6. Example N-glycan imaging mass spectrometry using a MALDI QTOF (timsTOF fleX, Bruker Scientific) on an FFPE triple negative breast cancer tissue with extensive tumor and necrotic regions. (A) An H&amp;E image of an invasive ductal carcinoma of the breast. Necrotic regions are the large pale vacuolar structures, largely surrounded by tumor. (B) An overlay image of three N-glycans: a necrotic region Hex5HexNAc4 with a single sialic acid (in green); a tumor localized Hex8HexNAc7Fuc polylactoseamine (in red); a tumor localized high mannose Hex9HexNAc2 (in blue). (C) Image segmentation of N-glycans (n   =   66) hierarchically clustered by localization and intensity on tissue. Hierarchical clusters from the image are in the right panel, with the number of spectra within each region represented by color. (D) A single heat map image for the necrotic glycan in panel (B)

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