Glycocalyx Gone Awry: Pathologic Cell Signaling during Endotheliopathy

The endothelial glycocalyx is a viscous mesh that lines the vascular lumen and serves a variety of functions in health and disease. This gelatinous matrix of proteoglycans and glycosaminoglycans is the critical contact point between the circulation and end organs; transmitting physical and biochemical signals through the endothelial surface layer to affect endothelial cell activity that subsequently impacts organ function. In this review we explore the roles of the major structural components of the glycocalyx in mediating cell signaling events during inflammatory-induced endotheliopathy. We also discuss proposed therapeutic strategies to attenuate glycocalyx disruption and promote its regeneration.


Introduction
The vascular endothelial glycocalyx (GCX) is the interface of blood-endothelial interactions and an active regulator of vascular tone and permeability. Overwhelming inflammatory states like trauma, sepsis, and burns precipitate a cascade of events that culminate in multiorgan dysfunction, primarily through endothelial cell activation and GCX disruption. Degradation of the GCX has been shown to increase vascular permeability, resulting in tissue edema; propagate local inflammation; and activate microvascular coagulation with resultant microthrombi and vascular occlusion.
Though much attention has been dedicated to understanding the pathophysiologic mechanisms leading to GCX disintegrity, our understanding of the precise endothelial cell signaling mechanisms affected by GCX degradation and their implications on vascular homeostasis remains incomplete. Our focus in this review will be on the known and posited downstream extra-and intracellular signaling events that contribute to vascular dysfunction after endothelial GCX disruption secondary to inflammation. We will also discuss the work that has been done to therapeutically target these mechanisms in an effort to restore vascular integrity.

Background
The GCX of the vascular endothelium is a matrix of luminal cellsurface proteoglycans (e.g., syndecan, glypican, versican, decorin, signaling to the intracellular environment through their highly conserved transmembrane domain6. Nuanced intracellular communications are dictated by the conserved C1 and C2 and variable V cytoplasmic domains of the Syn protein that associate with distinct enzymes and cytoskeletal elements [6]. The extracellular ectodomain of Syn is much more variable in its length compared to the cytoplasmic domain and predominantly expresses heparan sulfate, though post-translational substitutions with chondroitin sulfate moieties may also be expressed [6]. The diverse expression of HS lengths and sulfation patterns on Syn allows the GCX to serve as a large reservoir of associated plasma proteins and participate in a wide array of cell signaling pathways [7]. Glypicans only express an extracellular domain that is anchored to the endothelial cell membrane through a glycosylphosphatidylinositol anchor imbedded in lipid rafts and caveolae [8,9]. The remaining proteoglycans are expressed in the ESL as secreted molecules within the GCX and contribute significantly less its structure and function [10]. The most ubiquitously expressed GAGs in the endothelial GCX are heparan sulfate and hyaluronan [11]. Heparan sulfate (HS) is a highly variable GAG covalently bound to Syn and glypican on the endothelial cell surface and comprises over 50% of the GAG composition in the GCX. [11] The diversity of glucosamine and hexuronic acid (i.e., iduronic acid or glucuronic acid) polymerization and varied sulfation pattern allows the HS molecule to associate with a host of plasma proteins with specificity [12]. Imbedded within the HSPG mesh of the GCX are cytokines and chemokines that form gradients for endothelial cell signaling and leukocyte chemotaxis [7]. Additionally, the interaction of growth factors with HS on parent HSPGs governs the activation of the parent cell surface growth factor receptors6. Hyaluronan, or hyaluronic acid (HA), exists as a high molecular weight (HMW) GAG (over 1MDa) attached to the ESL through interactions with CD44 and other GAGs11. Its function is related to its uniquely non-sulfated, very large structure that provides both a lubricating and structurestabilizing role in the GCX. As a relative proportion, the expression of chondroitin, dermatan, and keratan sulfates is approximately four times less than that of heparan sulfate [11]. Dermatan sulfate (also classified as chondroitin sulfate type B) and keratan sulfate are primarily bound to secreted proteoglycans and exist more prevalently in the extracellular matrix of endothelial cells [11].
These lesser understood GAGs appear to contribute little to ESL function.
In health, the intact GCX serves several vital homeostatic functions. The negatively charged, highly proteinaceous composition of the GCX provides an electrostatic and physical barrier to circulating proteins to limit their extravasation and concomitantly limit the flow of circulating water to the extravascular space via modified Starling forces [13]. Endothelial cell surface receptors are buried within this luminal matrix and rely on proteoglycans and GAGs as co-receptors for cell signal activation [14]. Circulating cell interactions with the endothelium are regulated by cytokine and chemokine gradients within the GCX and by the various cell adhesion molecules hidden within the GCX [15,16]. The ESL also contains expressed cofactors for coagulation homeostasis, including antithrombin III (ATIII), thrombomodulin, tissue factor, and tissue factor pathway inhibitor. Thus, the GCX serves as a key touchpoint between and regulator of systemic inflammation and coagulation.
Moreover, the gelatinous-like nature of the GCX transmits shear stress and hydrostatic pressure to the endothelial cells, resulting in endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) regulation that coordinates vascular tone and permeability [17].

Role of Heparan Sulfate
In adults with respiratory failure or sepsis, circulating HS levels are significantly elevated compared to controls and correlate with illness severity [18,19]. Plasma HS levels are also higher in patients after traumatic injury [20] or major vascular procedures that induce systemic ischemia/reperfusion [21]. The circulating HS is presumably shed from the endothelial GCX among other sources, suggesting a mechanistic linkage between inflammatory-mediated ESL perturbation and organ injury.
HS is selectively cleaved from its associated proteoglycan  Enzymatically active HPSE cleaves HS bound to HSPGs, which release HS fragments and stereotactically associated chemokines and growth factors (e.g., VEGF) into circulation and the local microenvironment. Soluble HS fragments are then capable of initiating receptor-mediated signaling by facilitating the interaction of the bound growth factor or cytokine with its respective receptor. As an example, HS facilitates VEGF-binding to the VEGF receptor leading to SRC-dependent phosphorylation of VE-cadherin. This increases VEcadherin internalization resulting in destabilization of intercellular junctions. Unbound HS fragments also serve as damage-associated molecular patterns recognized by TLR4, which results in NF-kB-mediated proinflammatory cytokine production. By contrast, unbound HS binds circulating histones, making them less available to propagate inflammation.

Role of Hyaluronan
Adults admitted to the intensive care unit with septic shock demonstrate elevated plasma HA levels compared to controls [19]. Schmidt et al. [18] similarly reported elevated circulating HA fragments in patients with lung injury. This group went on to describe that in two adult cohorts admitted for sepsis or acute respiratory distress syndrome, increased urinary levels of HA early in the presentation of systemic illness were predictive of the development of acute kidney injury and in-hospital mortality [36]. Taken together, these data suggest that soluble HA and its degradation products promote proinflammatory signaling that culminates in organ dysfunction.

Figure 2:
Role of soluble HA in inflammation. ESL-bound HA is degraded in inflammatory states by hyaluronidase or ROS released by activated neutrophils. Soluble LMW-HA binds via CD44 and/or TLR4 to produce a proinflammatory M1-type macrophage, whereas HMW-HA binds via CD44 to promote an anti-inflammatory M2 macrophage phenotype. LMW-HA also activates TRL4 signaling in endothelial cells that results in NF-kB activation and proinflammatory cytokine production. Additionally, LMW-HA can bind to and initiate CD44 signaling, which, via PKC , recruits RHAMM to the cell surface. CD44 signaling, both independently and in concert with RHAMM, terminates in cytokine upregulation (via PI3K/Akt/ NF-kB), cell proliferation and migration (via ERK1/2-MAP kinase) and cytoskeletal rearrangement (via Ras/Raf/Rac).

HA is shed from the GCX and further degraded by hyaluronidase
and/or reactive oxygen species released from activated leukocytes, resulting in the formation of low molecular weight (LMW)-HA [37]. LMW-HA fragments produce a proinflammatory cellular phenotype, demonstrated by the activation of the M1 phenotype in macrophages in vitro [38,39] and exacerbation of lung injury in in vivo models of airway hyperreactivity [40]. HA signaling appears to be fragment size-dependent as soluble HMW-HA promotes an anti-inflammatory M2 phenotype in macrophages [38]. Several endothelial cell surface receptors that recognize LMW-HA have been identified, including TLR4, CD44, and the receptor for HA-mediated motility (RHAMM) (Figure 2). Human dermal microvascular endothelial cells treated with LMW-HA upregulate proinflammatory cytokine production in a TLR4-dependent manner [41]. Evidence for this signaling pathway is supported by the protection of TLR4 knockout mice from LMW-HA-induced lung injury [42]. The interaction of HA with cell surface CD44 has been extensively studied, demonstrating a wide array of signaling cascades terminating in cytokine upregulation, cytoskeletal remodeling, and cell proliferation that can occur independently of or in concert with RHAMM [43]. The role of RHAMM in HA signaling appears to be more nuanced as its expression depends on various factors, including induction by CD44/protein kinase C activation [44]. After being expressed on the cell membrane, RHAMM binds with soluble LMW-HA, thereby activating the ERK1/2-MAP kinase pathway to promote cellular proliferation [45] and Ras/Raf/Rac to effect cytoskeletal rearrangement [43].

Role of Other Glycosaminoglycans
The other GAGs expressed in the endothelial GCX have received much less attention, limiting our understanding of their role in the inflammatory response. When shed, chondroitin sulfate appears to exhibit anti-inflammatory properties through histone-binding and NF-kB downregulation [46,47]. However, soluble chondroitin sulfates may also exhibit untoward antibacterial peptide inhibition in the setting of sepsis [48]. Dermatan sulfate may exhibit more tissue-specific local inflammatory regulation by mediating FGFRdependent cell proliferation and NF-kB activation with resultant increased ICAM-1 expression in and around epidermal wounds [49,50]. Keratan sulfate does not have a known role in inflammatory signaling at present.

Role of Surface Heparan Sulfate Proteoglycans
The HSPGs are the only known endothelial surface-anchored proteoglycans, making them integral in maintaining GCX integrity  [20,51,52] sepsis [48,53] and burns [54,55] and correlate with illness severity [48,51,54,56]. Though the glypicans have been less well studied, circulating glypican levels are elevated in the setting of sepsis and correlate with disease severity [57]. Adults with severe direct lung injury from pneumonia also demonstrate elevated levels of plasma glypican [58].
Mechanosensing of vascular shear stress to the endothelial cytoskeleton is primarily mediated through the HSPGs [59] (Figure   3). Syn1 and Syn4, in particular, directly influence cytoskeletal remodeling through syntenin and synectin linkages to the actin cytoskeleton [60][61][62][63][64]. As blood flow shear stress deforms the GCX, Syn transmits this torque to the actin cytoskeleton [65] via, in part, activation of RhoA GTPases [66]. Due to the intricate cytoplasmic connections of the cytoskeleton to adherens and gap junctions and intracellular organelles, the surface Syn-mechanotransduction becomes "decentralized" and propagates throughout the cytoplasm [67]. The resultant cytoskeletal reorganization influences cellular shape and distribution along the vasculature, inter-and intracellular molecular transport, cell-cell adhesions, and nuclear activation [65,67]. Syn4 also activates protein kinase Cα in a calcium-independent fashion to maintain proper cytoskeletal stress fiber alignment in response to blood flow [60,68]. Moreover, Syn4, through its interaction with Rho, activates focal adhesion kinase to maintain focal adhesion integrity [69]. Thus, loss of Syn expression from the ESL would be expected to culminate in destabilized cytoskeletal arrangement with poor endothelial responsiveness to changes in surface shear as demonstrated by Baeyens et al. [70] Degradation of the HSPGs appears to also significantly alter the endothelium's ability to regulate vasoreactivity to vascular shear stress by disrupting eNOS activation. Voyvodic et al. [66] have shown that loss of Syn1 from the GCX suppresses the endothelial cell's ability to activate the PI3/Akt pathway in response to luminal shear, resulting in decreased phosphorylated Akt. Dimmeler et al. [71] have demonstrated that eNOS is activated in response to Akt phosphorylation. Taken together, the results of these studies suggest that loss of Syn1 expression from the GCX may result in decoupling of eNOS activation from vascular blood flow. HS trimming by heparinase III, a bacterial enzyme with nonspecific HS-degrading activity at the ESL, has also been shown to abrogate shear-induced eNOS activation [59,72]. Likewise, glypican influences caveolae protein 1 expression that subsequently regulates eNOS expression in caveolae [73][74][75]. Thus, deranged glypican expression suppresses eNOS activity in response to shear via decreased eNOS expression [9,76]. Interestingly, HA also participates in mechanosignaling to influence eNOS activation [77,78]. Thus, removal of HSPGs and HA impairs shear-induced vasoactivity of the endothelial cell by attenuating eNOS expression and activation.
HSPGs protect the endothelium from leukocytes and platelets by shielding cell adhesion molecules and glycoproteins used by these circulating cells for endothelial attachment [79,80]. HPSE-mediated removal of HS from Syn opens the proteoglycan up to the enzymatic activity of MMPs [81][82][83], thereby exposing P-and E-selectins and VCAM-1, PECAM-1, and ICAM-1. Now available, circulating leukocytes and platelets adhere to these cell adhesion molecules and become activated [4,80,84,85]. Indeed, the result of loss of Syn1 endothelial surface expression has been shown to promote a proinflammatory phenotype that is abrogated by reexpression of Syn1 [66]. Similarly, treatment of various endothelial cell lines with heparinase III has been shown to significantly increase leukocyte adhesion with resultant tissue injury [4,86]. Release of chemokines from the cell surface during GCX degradation into the local microenvironment may serve to attract leukocytes to and through the endothelium. Conversely, chemokine release from the ESL may also act as a mechanism to reduce leukocyte attachment to the endothelium [87]. Furthermore, like HSPGs, cell adhesion molecules may undergo enzymatic cleavage by MMPs [88]. Shapiro et al. [89] found plasma levels of the soluble form of several cell adhesion molecules (e.g., sE-selectin, sICAM-1, and sVCAM-1) to be significantly elevated in adults presenting to the emergency department with sepsis. While the purpose for cell adhesion molecule cleavage is not entirely clear, it may provide a protective mechanism for the endothelium to limit leukocyte attachment and activation or it may serve as an essential step in the active invasion of the leukocyte through the endothelium by dissociating from the ESL. Thus, the degree to which the GCX prevents or aides in leukocyte chemotaxis, attachment, and activation during inflammatory-related ESL disruption may be dictated by the degree of remaining HS and cell adhesion molecule expression after GCX degradation [90]. The cytoplasmic domain of Syn is associated with the actin cytoskeletal network via linkages with syntenin and synectin, which allows for propagation of mechanotransduction signaling from the cellular surface to intercellular junctions and throughout the cytoplasm. In response to shear stress, Syn initiates cytoskeletal alignment and focal adhesion formation via activation of RhoA. In some cell types, Syn1 may also regulate shear stress-induced activation of PI3K/Akt signaling. Glypican, HS, and HA mediate cellular activation of eNOS and NO production in response to shear stress via associations with caveolae/lipid raft regions that are enriched with signaling molecules (e.g., inactive eNOS). Loss of any or all of these GCX constituents impairs the endothelial cell response to intra-luminal forces.
FGF2-FGFR signaling is heavily influenced by Syn4 stabilization of the growth factor-receptor complex to affect a multitude of complex intracellular pathways that result in maintenance of HS expression in the GCX and stabilization of endothelial cell-cell adhesions [92].
Constitutive expression of Syn4 also contributes to the tightly controlled release of angiopoietin-2 (Agpt-2) from the endothelium [93]. Agpt-2 is a cytokine that antagonizes the endothelial cell surface Tie2 receptor, resulting in destabilization of cell-cell junctions [94]. Thus, loss of Syn-4 ESL expression may contribute to dysregulated FGFR signaling and Agpt-2 release that would result in increased endothelial permeability and impaired GCX restoration. The role that glypican disruption plays in endothelial cell receptor-mediated signaling during inflammation remains to be studied [95].

Therapeutic Strategies Targeting GCX Restoration
As our knowledge of the signaling mechanisms influenced by the GCX is continually growing, so too is the development of novel strategies to (1) prevent GCX damage, (2) inhibit the downstream detrimental signaling cascades initiated by GCX shedding, and/or Well-intentioned goal-directed clinical practices (e.g., early highvolume crystalloid resuscitation for hemorrhagic or septic shock) may actually worsen clinical outcomes, [96,97] in part through GCX perturbation [98,99]. The use of fresh frozen plasma (FFP) as a resuscitation fluid has received much attention due to its ability to upregulate GCX synthesis, promote repair of EC junctions, and restore microvascular function after hemorrhagic shock [98,[100][101][102]. More recently, the GCX-protective benefits of FFP resuscitation have been attributed to its adiponectin [103], ATIII [104][105][106].
Other therapies that demonstrate GCX-protection include hydrocortisone104 via mitigation of TNFα-induced endothelial activation; dexamethasone [107] and sphingosine-1-phosphate8 by downregulating endothelial cell MMP expression; doxycycline [108] via MMP inhibition; and heparin/heparan sulfate mimetics derivatives [4,109,110] via HPSE inhibition. To prevent aberrant downstream signaling events after GCX injury, purified HMW-HA may be administered systemically [111]. Lastly, ESL regeneration may be promoted by FFP as described above; sphingosine-1phosphate by increasing ESL HS expression [112]; sulodexide [113,114] by re-coating the ESL with highly purified GAGs; and FGF-receptor agonists to promote exostosin-1 expression and HS synthesis [115]. Although promising, these therapies remain theoretical at present and require further study with prospective randomized clinical trials [116]. There is compelling evidence to suggest that strategies targeting the prevention of GCX damage and the restoration of its integrity will improve vascular function, which has the potential to meaningfully improve clinical outcomes. Given the progress in the field of endothelial GCX research over the last several years, we anticipate significant translational developments of therapies aimed at protecting the GCX in the years to come.