Joim.12331.pdf

Review This paper contains Video

doi: 10.1111/joim.12331

Endothelial barrier dysfunction in septic shock S. M. Opal1 & T. van der Poll2 From the 1Infectious Disease Division, Alpert Medical School of Brown University, Pawtucket, RI, USA; and 2Academic Medical Center, Division of Infectious Diseases & The Center of Experimental and Molecular Medicine, University of Amsterdam, Amsterdam, the Netherlands

Abstract. Opal SM, van der Poll T (Alpert Medical School of Brown University, Pawtucket, RI, USA; University of Amsterdam, Amsterdam; the Netherlands). Endothelial barrier dysfunction in septic shock (Review). J Intern Med 2015; 277: 277–293. The endothelium provides an essential and selective membrane barrier that regulates the movement of water, solutes, gases, macromolecules and the cellular elements of the blood from the tissue compartment in health and disease. Its structure and continuous function is essential for life for all vertebrate organisms. Recent evidence indicates that the endothelial surface does not have a passive role in systemic inflammatory states such as septic shock. In fact, endothelial cells are in dynamic equilibrium with a myriad of inflammatory mediators and elements of the

Introduction Sepsis, severe sepsis and septic shock comprise a spectrum of increasingly common, potentially lethal, yet poorly understood clinical syndromes. The rising incidence is well documented and thought to be attributable to the ageing of the population, the prevalence of immunocompromised patients and implantable medical devices and the progressive increase in antimicrobial resistance among common bacterial pathogens [1–5]. Developing new therapeutic agents to manage patients with sepsis has proven difficult with a large number of failed clinical trials [6–8]. Despite the repeated failure to demonstrate survival benefits for a number of promising novel therapeutic agents for the treatment of sepsis in Phase III clinical trials, the news regarding clinical outcomes in patients with sepsis is not all bad [6, 7, 9]. The mortality rate is certainly improving in many clinical studies worldwide, which is primarily a reflection of improved supportive care and early diagnosis and treatment protocols [10–13]. Additionally, substantial progress has been made in

innate immune and coagulation systems to orchestrate the host response in sepsis. The barrier function of the endothelial surface is almost uniformly impaired in septic shock, and it is likely that this contributes to adverse outcomes. In this review, we will highlight recent advances in the understanding of the signalling events that regulate endothelial function and molecular events that induce endothelial dysfunction in sepsis. Endothelial barrier repair strategies as a treatment for sepsis include modulation of C5a, high-mobility group box 1 and VEGF receptor 2; stimulation of angiopoietin-1, sphingosine 1 phosphate receptor 1 and Slit; and a number of other innovative approaches. Keywords: endothelial barrier, endothelial junctions, protease-activated receptors, sepsis, sepsis-induced immunosuppression, septic shock.

understanding the basic pathophysiology of sepsis from the molecular level to the level of organ communication and systems integration [14–24]. Currently, the nature of sepsis is viewed as a fundamental disintegration of systems controls from intercellular signalling networks to neuroendocrine and immune regulatory mechanisms during the time of systemic injury and invasive microbial stress. Failure to maintain or reconstitute homeostasis after a major systemic injury ultimately determines outcome in sepsis [7, 19, 22]. The immediate threat in sepsis is invasive infection, and the need to activate immune defences to clear the pathogen before irreparable damage occurs due to the microorganism and its toxins. However, in the process of eliminating the pathogen, the systemic host response to generalized infection can lead to collateral damage to normal tissues [4]. It is remarkable that many of the same pathways, pattern recognition receptors and innate host responses seen in sepsis are also observed in other forms of severe injury and systemic inflammation without concomitant infection [2, 4, 22].

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The long-term consequences of sepsis can be profound and disabling. These consequences are increasingly recognized as a condition now referred to as persistent critical illness (PCI). PCI may persist for months or even years and is characterized by prolonged lengths of stay in the intensive care unit, persistent organ dysfunction, dysfunctional host response to repeat infections, slow or even permanent cognitive decline and losses of overall sense of well-being and function in society [25–27]. Sepsis is frequently followed by a reduced duration of healthy life expectancy, even if the patient’s overall lifespan is preserved. In this review, we will focus on recent findings regarding the basic elements that drive the septic process at the level of cellular communication, the coagulation system and endothelial barrier function. Of note, other research priorities are clearly important in understanding the basic mechanisms that underlie the pathophysiology of sepsis, including disordered immune function [28–30], neuroendocrine [31–33], nutritional [34], immune metabolism [18], mitochondrial sparing [23, 35, 36] and systems integration studies [14, 19]. The importance of immune dysfunction and elements of immune depression in sepsis is increasingly being recognized, and they might prove to be suitable targets for therapeutic intervention [28–30]. Loss of adaptive immune function and downregulation of the host innate immune response have been well described and cause adverse consequences. For example, opportunistic viral infections are reactivated in the majority of patients with severe sepsis, and these infections can cause clinically significant conditions such as systemic cytomegalovirus infection and herpes simplex activation [37]. What is not clear is whether sepsis-induced immune suppression is a necessary compensatory mechanism to regulate the systemic septic host response or a potentially treatable, pathological state of immune suppression increasing the risk of superinfections in patients [38, 39]. This question can only be answered by carefully controlled clinical trials in well-characterized patient populations. We wish to highlight the loss of cellular barriers and its consequences at the level of the endothelial membrane to make our point about loss of function of vascular integrity in sepsis. In a recent review, Deutschman and Tracey emphasized that loss of cell membrane barrier function is a general finding 278

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in septic patients [7]. The histopathological findings of autopsy examinations of patients who have died as a result of multiorgan dysfunction due to sepsis (and in experimental animal studies of sepsis) are often remarkably normal appearing, characterized primarily by mild tissue oedema in the intracellular and extravascular interstitium. These anatomical findings seem incongruous with the clinical findings in such patients with evidence of acute kidney injury, cholestatic jaundice, acute myocardial dysfunction and similar dysfunctions in other organs. Despite the relative preservation of tissue morphology, tissue function is often markedly impaired. Cardiac myocytes stop contracting normally, alveoli cease to maintain the air–liquid barrier interface in lung tissue, hepatocytes no longer secrete bilirubin, endothelial cells retract, become permeable to macromolecules and lose their anti-adhesive and anticoagulant surface characteristics, and so on. The entire process seems to be a manifestation of poor cellular and tissue barrier function, loss of specialized tissue actions and an acquired form of cellular hibernation. Tissues stop generating variability in the integrated circuitry and stop generating cycles of communication within and between tissues. The loss of specialized cell function and barrier function has been noted by many investigators, suggesting that this constitutes a common host response to sepsis and other forms of critical illness [7, 15, 22]. Here, we will review the evidence that loss of barrier function in general, and endothelial barrier function in particular, is central to the pathogenesis of sepsis and highly integrated into the host systemic inflammatory and coagulopathic response. A number of authors have recently developed this concept of the aberrant and dysfunctional endothelial barrier as the central pathophysiological process in septic shock [40–44]. We will also discuss how endothelial barriers are influenced by coagulation factors and elements of the innate immune response, and review novel findings concerning monocyte/macrophage–endothelial interactions and signalling in sepsis. The vascular endothelium and haemostasis Endothelial cells are at the interface between inflammation and coagulation in sepsis [43] (Fig. 1). Dysfunction of the endothelium has an important role in the disturbance of the haemostatic balance in sepsis. Moreover, the endothelium

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Coagulation ↑

Microcirculation

Tissue factor ↑

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Anticoagulation ↓

TFPI ↓ Antithrombin↓

MyD88-ARNO-ARF6

Endothelial cell

TM ↓ EPCR ↓ Protein C (↓)

Tie2 Angiopoietin-2 ↑

Robo4

Monocyte APC ↓

Blood pressure ↓ Fibrinolysis ↓ NETs with trapped platelets

Neutrophil

RBC deformability↓

PAI-1 ↑

APC ↓ - Thrombin ↑

Thrombosis

PAR1

VE-cadherin↓ Tight junctions ↓

Cell death Cell shrinkage

S1P3/S1P1 ↑

Tissue hypoperfusion

Loss of barrier function

Tissue

Capillary leak Interstitial oedema

Tissue oxygenation ↓ Organ failure

Fig. 1 Dysfunction of the vascular endothelium in severe sepsis. Sepsis is associated with microvascular thrombosis due to concurrent activation of coagulation (mediated by tissue factor) and impairment of anticoagulant mechanisms as a consequence of reduced activity of endogenous anticoagulant pathways mediated by activated protein C (APC), antithrombin and tissue factor pathway inhibitor (TFPI) plus impaired fibrinolysis due to enhanced release of plasminogen activator inhibitor type I (PAI-1). The capacity to generate APC is impaired at least in part due to reduced expression of the endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). Thrombus formation is further facilitated by neutrophil extracellular traps (NETs) released from dying neutrophils. Thrombus formation results in tissue hypoperfusion, which is aggravated by hypotension and reduced red blood cell (RBC) deformability. Tissue oxygenation is further impaired by loss of barrier function of the endothelium due to loss of vascular endothelial (VE)-cadherin function, alterations in the endothelial cytoskeleton, high angiopoietin-2 levels (which interact with the endothelial cell receptor Tie2), and activation of Myd88–ARNO–ARF6 signalling. ARF6 is adenosine diphosphate-ribosylation factor 6; ARNO is ARF nucleotide-binding site opener. A disturbed balance between sphingosine 1 phosphate receptor (S1P)1 and S1P3 within the vascular wall at least in part due to preferential induction of S1P3 via protease-activated receptor 1 (PAR1) secondary to a reduced APC/thrombin ratio. Robo4 is an endothelial cell receptor that protects barrier function.

is involved in all three major pathogenetic pathways associated with coagulopathy in sepsis: tissue factor (TF)-mediated thrombin generation, dysfunctional anticoagulation and impaired fibrinolysis. TF is the main initiator of coagulation activation in sepsis. It is clear that monocytes and macrophages are major sources of TF in severe sepsis; however, endothelial cells also contribute to a considerable extent [44]. Endothelial cells activated by bacterial products or proinflammatory cytokines such as tumour necrosis factor (TNF)-a or interleukin (IL)1b express TF at their surface. Expression of TF by endothelial cells in vivo is restricted to certain organs and vascular beds [42]. TF binds to and activates clotting factor VII, which via factor X

results in the generation of thrombin and fibrin. The pivotal role of TF in sepsis-induced coagulation has been established in early clinical studies in humans and primate models in which strategies that prevent the activation of the TF–factor VIIa pathway abrogated the activation of the common pathway of coagulation elicited by administration of lipopolysaccharide (LPS) or bacteria [45]. In addition, in lethal sepsis models in baboons, TF inhibition prevented multiple organ failure and mortality [46, 47]. Similarly, in genetically deficient mice with an almost complete lack of TF, coagulation, inflammation and mortality induced by injection of high-dose LPS were reduced relative to control mice [48]. In addition, in its cell-associated form, TF can reside in microparticles (MPs) shed from haematopoietic and endothelial cells. MPs ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2015, 277; 277–293

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have been implicated in the activation of both coagulation and inflammation in sepsis [49]. Furthermore, upon stimulation with proinflammatory cytokines, endothelial cells are able to release alternatively spliced TF (lacking exon 5) which circulates in a soluble form and may exert procoagulant activity [50]. Coagulation is regulated by three main anticoagulant mechanisms: antithrombin, the protein C system and TF pathway inhibitor (TFPI). Sepsis is associated with impaired function of all three pathways, primarily as a consequence of endothelial dysfunction [44]. Resting endothelial cells generate activated protein C (APC) on the cell surface, produce tissue-type plasminogen activator to stimulate fibrinolysis and impede thrombin formation and platelet adhesion. Antithrombin is the main inhibitor of thrombin and activated (a) factor X (Xa). Under physiological conditions, glycosaminoglycans present on the vessel wall support antithrombin-mediated inhibition of thrombin and other clotting enzymes. During sepsis, antithrombin concentrations are strongly reduced due to impaired production, enhanced degradation (at least in part by elastase from activated neutrophils) and consumption caused by sustained thrombin generation [45]. Antithrombin function is further compromised by the decreased production of glycosaminoglycans on the endothelial surface, which is mediated by proinflammatory cytokines [43]. TFPI is the main inhibitor of the TF–factor VIIa complex, which is predominantly expressed by endothelial cells [51]. TFPI binds with high affinity both to TF–factor VIIa and to factor Xa present in plasma and on endothelial cells. Under normal conditions, TFPI is attached to the endothelium via proteoglycans, which facilitates its TF–factor VIIa– factor X-inhibiting properties on the endothelial surface [52]. TFPI function is inhibited in sepsis by the reduced synthesis of glycosaminoglycans on the endothelial surfaces. The importance of TFPI as an endogenous anticoagulant in sepsis is illustrated by the fact that rabbits became more susceptible to LPS-induced disseminated intravascular coagulation (DIC) and the generalized Shwartzman reaction following inhibition of TFPI [53, 54]. Administration of recombinant TFPI dosedependently inhibited LPS-induced thrombin generation in humans [55]. Pharmacological doses of TFPI can prevent mortality during systemic infection and inflammation, indicating that high TFPI 280

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concentrations are able to modulate TF-mediated coagulation [46, 47]. The vascular endothelium serves an important role in the protein C system, which represents a crucial endogenous anticoagulant mechanism by virtue of the ability of APC to proteolytically inactivate coagulation cofactors Va and VIIIa. APC is formed from protein C when thrombin binds to the thrombomodulin receptor present on endothelial cells. The activation of protein C to APC by thrombomodulin-bound thrombin is augmented by the presence of the endothelial protein C receptor (EPCR). During sepsis, the protein C system is impaired as a result of several factors, most notably decreased synthesis and increased consumption of protein C and decreased activation of protein C due to reduced expression of thrombomodulin and EPCR on endothelial cells [45]. The anticoagulant strength of the protein C system has been demonstrated in many preclinical settings [56]. In a landmark study conducted in the 1980s, Taylor and colleagues demonstrated that infusion of APC into septic baboons prevented DIC and death and that inhibition of protein C activation aggravated the response to Escherichia coli and transformed a sublethal effect into a lethal DICassociated state [57]. In accordance with an important regulatory function of the endogenous protein C system in sepsis, treatment of bacteraemic baboons with an anti-EPCR monoclonal antibody exacerbated a sublethal E. coli infection leading to lethal sepsis with massive activation of coagulation [58]. The anticoagulant effects of APC in haemostasis may be enhanced by its capacity to inhibit fibrinolysis by suppressing the function of two fibrinolysis inhibitors: thrombin activatable fibrinolytic inhibitor (TAFI) and plasminogen activator inhibitor type I (PAI-1) [56]. Adhesion of cells to injured endothelium relies on adhesive proteins, such as von Willebrand factor. Monomers of von Willebrand factor (280 kDa) can be linked by disulfide bonds to form ultra-large multimers, with a molecular mass of up to 106 Da [59]. The assembly of von Willebrand factor multimers occurs in endothelial cells, where they are stored in Weibel–Palade bodies. Large multimers of von Willebrand factor, which are released upon endothelial cell perturbation, can bind more efficiently to platelet glycoprotein Iba. In normal haemostasis, large von Willebrand factor multimers

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are cleaved by a protease termed a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13); this process is stimulated under circumstances of high shear stress. Physiologically, von Willebrand factor acts to stabilize the adhesion of platelets at sites of vascular injury. Sepsis is associated with a relative deficiency of ADAMTS13, resulting in increased levels of ultra-large von Willebrand factor multimers, which facilitate platelet adhesion to injured endothelium [60]. It has been suggested that the resulting thrombotic microangiopathy has an important role in multiple organ dysfunction in sepsis [61]. Activated platelets form a strong link between the processes of primary and secondary haemostasis by providing the phospholipid layer necessary for the assembly of activated coagulation factor complexes [62]. Disruption of the vascular integrity in sepsis results in the adhesion of platelets at sites of injury, where they can contribute to endothelial cell activation through several mechanisms. For example, stimulation of glycoprotein IIb/IIIa on platelets enhances CD40 ligand expression on the platelet membrane, which activates endothelial cells to express adhesion molecules and TF [63, 64]. Expression of P-selectin on the platelet membrane mediates the adherence of platelets to leucocytes and endothelial cells and enhances the expression of TF on monocytes [65]. Vascular inflammation and coagulation are amplified by the release of so-called neutrophil extracellular traps (NETs) by neutrophils. NETs are highly charged mixtures of DNA and nuclear proteins together with serine proteases such as elastase, cathepsin G and calprotectin [66]. NETs function to entrap pathogens (an action that is facilitated by platelets) and are designed to mediate swift elimination of invasive microorganisms. However, as for many components of innate immunity, NETs can also contribute to collateral tissue damage [67, 68]. Indeed, NETs are procoagulant by promoting adhesion, activation and aggregation of platelets and by activating the contact system, leading to the release of bradykinin and activation of the intrinsic pathway of coagulation [68]. Moreover, by promoting platelet and red blood cell adhesion, and by engagement of clotting factors, NETs can provide a scaffold for thrombus formation. Additionally, NETs can induce endothelial cell death, an effect that is likely to be mediated by NET-associated proteases or cationic proteins such as defensins

Review: Endothelial dysfunction in sepsis

and histones [69]. Finally, NETs generated as a consequence of platelet–neutrophil interactions have been implicated in tissue damage in sepsis [67]. Protease-activated receptors and endothelial barrier function Thrombin and an array of circulating serine proteases can cleave a set of ubiquitously expressed, seven-transmembrane receptors known as protease-activated receptors (PARs) [43, 44]. Four such receptors (PAR1–4) are found in humans and can either disrupt or protect endothelial barrier function, depending on which G-protein-linked intracellular signalling pathway is activated. PARs are unusual receptors as the ectodomain contains the internal, sequestered ligand that, through feedback, activates its own receptor, but only if the Nterminus of the ectodomain is removed by partial proteolysis by serine proteases such as thrombin [43–45]. Thrombin binds to PAR1 expressed on endothelial cells in the early phase of sepsis, contributes to endothelial dysfunction by G12/13 Rho-dependent cytoskeletal derangements in endothelial cells and induces endothelial cell contraction and rounding [70, 71]. Endothelial cell contraction destabilizes cell-to-cell contacts, causing an increase in vascular permeability, which facilitates the passage of large molecules (albumin and other plasma proteins) and leucocytes from the blood into the subendothelial compartment (see Fig. 2). Gaps in the endothelial barrier also expose the fluid compartment of blood to the basement membrane and vessel adventitia with abundant TF for clot initiation and collagen fibres for von Willebrand factor to polymerize and for binding platelets. It is interesting that thrombin-induced stimulation of endothelial cells by PAR1 activation can be deleterious to barrier function or beneficial to endothelial function depending on the stage of progression of sepsis and severity of the disease state [72]. Over time, thrombin linked to PAR1 can transactivate PAR2 into a PAR1–PAR2 heterodimer, which has a protective role in endothelial barrier function and survival in sepsis models. This ‘role reversal’ for PAR1 signalling from endothelial barrier disruption to barrier protection is mediated by PAR1 to PAR2 combined signalling that switches coupling of the GTPase RhoA intracellular barrier disruptive pathway to an alternative PAR2-GiRac1-mediated intracellular signalling pathway resulting in actin polymerization and improved ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2015, 277; 277–293

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Tight endothelial barrier

Dysfunctional barrier C’5a, PAF, NO Bradykinin VEGF receptor 2 Thrombin, FXa, MMP HMGB1

Activated protein C Atrial natriuretic peptide Robo4-Silt2N Ang1-Tie2 Protease inhibitors PAR1-2

Review: Endothelial dysfunction in sepsis

PAR1

VE-cadherin

S1P3

S1P1 RhoA

Rac1 TF

Tie2 Tie2 Ang-1 TF

Collagen fibres

TF

TF

Actin polymer cytoskeleton

Macrophage–endothelial cell interactions TF

Actin breakdown

Fig. 2 Endothelial barrier function and dysfunction in health and disease. The left panel shows the factors that contribute to tight junctions, with well-formed and functional endothelial cells covering the microvasculature. The right panel highlights those molecular signals that contribute to loss of barrier function and endothelial cell rounding and retraction. Breakdown of tight junctions is accompanied by loss of intravascular proteins to the interstitial spaces, procoagulant and proinflammatory processes, and loss of barrier integrity. Ang, angiopoietin; Tie2, tyrosine kinase with immunoglobulin-like and epithelial growth factor-like domains 2; VE, vascular endothelium; PAR1–2, protease-activated receptor type 1 and 2 heterodimer; Rac1–Cell division control protein; TF, tissue factor; S1P, sphingosine 1 phosphate receptor; C’, complement; PAF, platelet-activating factor; NO, nitric oxide; FXa, activated factor X; MMP, matrix metalloprotease; VEGF receptor 2, vascular endothelial growth factor receptor-2; HMGB1, high-mobility group box 1; RhoA, GTPase of the Ras homolog gene family.

barrier function. This dual signalling system might be amenable to therapeutic intervention with agents that target specific PARs at specific times during the progression of sepsis (see Table 1 and discussion below) [72–74]. Recently, matrix metalloprotease (MMP)1 has been detected at high levels in the circulation in patients with severe sepsis/septic shock (18-fold higher than normal blood levels) [75]. This might have significant prognostic and therapeutic implications. MMP1 efficiently cleaved PAR1 on endothelial cells but, in contrast to thrombin with dual signalling ability, MMP1–PAR1 signalling mediates only barrier disruption [75]. Endothelial-derived MMP1 activates noncanonical PAR1 signalling and is associated with poor outcomes in experimental models of septic shock [76, 77]. MMP1 cleaves and activates PAR1 at a novel D39–P40 site, which leads

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to biased-barrier breakdown signalling in endothelial cell membranes [76, 77]. This cleavage site is located two amino acids proximal to the N-terminus site for thrombin cleavage at position R41–S42. The MMP1 cleavage site generates a slightly longer, tethered peptide ligand which mediates barrier disruption upon PAR1 activation [72, 77].

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The occurrence of crosstalk between myeloid and endothelial cells has been well known for many years as endothelial signals are critical for targeting neutrophils and circulating monocytes from the vascular lumen to endothelial surfaces expressing adhesion molecules in areas of acute inflammation [65]. Activated endothelial cells secrete IL-8, monocyte chemoattractant protein-1 and macrophage inhibitory protein-1 to attract monocytes to inflammatory foci. Intravascular, adherent, activated monocytes express large amounts of TF to propagate procoagulant activity at the site of infection or injury. However, the extent to which tissue macrophages are involved in angiogenesis, tissue repair and maintenance of endothelial barrier function is just beginning to be understood. Macrophages are initially activated after tissue injury by ischaemic necrosis of mesenchymal cells in an IL-1a-dependent process. IL-1a primarily exists as an uncleaved but biologically active precursor which is located within the intracellular space and nucleus where it regulates transcription [78]. During necrotic, but not apoptotic, cell death, the IL-1a precursor is released from intracellular stores within dying mesenchymal cells and binds to its type 1 IL-1 receptor expressed on adjacent tissue macrophages. By contrast, during apoptosis, IL-1a precursor remains tightly bound to the chromatin where it is then digested during the apoptotic nuclear fragmentation process without IL-1a release [79]. Necrotic cell release of IL-1a precursor is rapidly followed by inflammasome assembly within the macrophage cytosol, resulting in caspase-1 synthesis, IL-1b processing and extracellular secretion with marked upregulation of IL-1b -induced chemokine, cytokine and adhesion molecule expression on nearby endothelial cells [79, 80]. Recruitment of circulating neutrophils and monocytes activates inducible nitric oxide synthase with rapid efflux of nitric oxide followed by vasodilatation, opening of endothelial gaps and loss of endothelial barrier function (Fig. 2) [78].

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Table 1 Endothelial barrier disrupters and promoters Barrier disrupters

Barrier promoters

Molecule

Mechanism

Molecule

Mechanism

C’ components

Vasodilators, increase

C’ regulators and

Block anaphylatoxin

C3a, C5a Bradykinin

vascular permeability Vasodilator, increases

carboxy-peptidases

actions, degrade C3a and C5a

Carboxy-peptidases

Degrade bradykinin

PAF-acetyl hydrolase

Degrades PAF in the

Anti-inflammatory

Limit expression of

permeability PAF

Vasodilator, increases permeability

Proinflammatory cytokines

Increase surface expression of adhesins,

circulation cytokines

inflammatory cytokines

PMN–EC interaction Ang-2

Inhibitor of Ang-1, pro-apoptotic

Ang-1

Inhibits NF-jB signalling,

Tie1

Specific inhibitor of Tie 2

Tie2

Tyrosine kinase receptor

RhoA GTPase

Breaks down actin cytoskeleton,

Rac1

Stabilizes actin cytoskeleton

PAR1 signals

Pro-apoptotic, activate RhoA

PAR1–PAR2 transactivation

Anti-apoptotic, promotes Rac1

MMPs

Activate PAR-1 barrier disruption

Protease inhibitors

Limit activity of MMPs

S1P1

Membrane stabilizer,

inhibits apoptosis interaction with Ang-1

for Ang-1, anti-apoptotic

internalizes VE-cadherin

and RhoA S1P3

Activates RhoA, de-polymerizes actin cytoskeleton

promotes VE-cadherins, actin polymerization

Vascular endothelial growth factor 2

Activates Src kinase to

Atrial natriuretic peptide

phosphorylate and internalize

Activates Rac1, antiinflammatory, limits NF-jB

VE-cadherin b-arrestin-mediated endocytosis

Internalizes phosphorylated

Slit–Robo4

Inhibits VE-cadherin

VE-cadherin, impairs

phosphorylation by

endothelial barrier

p120-catenin, blocks apoptosis

HMGB1

Breaks down VE-cadherin,

HMGB1 mAb

Inhibits HMGB1-dependent

promotes cytokine and

alteration of endothelial

chemokine generation

barrier function

Ang, angiopoietin; S1P, sphingosine 1 phosphate receptor; NF-jB, nuclear factor kappa light-chain enhancer of B cells; VE, vascular endothelial; C’, complement; PMN, polymorphonuclear cell; PAF, platelet-activating factor; EC, endothelial cell; Tie 1 and Tie 2, tyrosine kinase with immunoglobulin-like and epithelial growth factor-like domains; GTPase, guanosine triphosphatase; RhoA, GTPase of the Ras homolog gene family; Rac1, a subfamily of GTPases; PAR, proteaseactivated receptor; MMP, matrix metalloprotease; Src kinase, sarcoma-related nonreceptor protein kinase; HMGB1, highmobility group box 1; mAb, monoclonal antibody.

Extra-luminal macrophages support endothelial cell growth, limit apoptosis and remodel the extracellular matrix via expression of MMPs to support endothelial membrane structure and function [81–83]. Tissue macrophages signal to endothelial cells by one of three mechanisms: (i)

synthesis and release of soluble growth factors into the microenvironment, (ii) local release of macrophage exosomes and (iii) direct cell-to-cell contact with endothelial cells (Fig. 3). Vascular endothelial growth factor A, colony-stimulating factor 1, IL-1b and TNF-a are secreted by ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2015, 277; 277–293

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Endothelial barrier

Activated monocytes

Capillary lumen

PSGL1

MIP-1 MCP1 IL- 8

TF

HMGB1 TF

TF

P-selectin

Exosomes

Soluble cytokines

RAGE TLR4

TNF MCP1 IL-1α IL-1β

Ang2 Tie2 Direct cell-tocell contact

Tissue macrophages activating ECs Fig. 3 Interactions between tissue macrophages in the perivascular space, circulating monocytes and endothelial cell surfaces. High-mobility group box 1 (HMGB1) is a late cytokine-like mediator in sepsis that increases endothelial permeability and induces inflammatory cytokine synthesis by endothelial cells (ECs). PSGL1, P (platelet)-selectin glycopeptide ligand 1; MIP1, macrophage inhibitory protein 1; MCP1, monocyte chemoattractant protein 1; IL, interleukin; TNF, tumour necrosis factor; TF, tissue factor; TLR4, Toll-like receptor 4; RAGE, receptor for advanced glycation end products.

activated tissue macrophages. These cytokines and growth factors bind to their cognate receptors expressed on endothelial cells to support growth and to promote expression on vascular endothelial surfaces of adhesion molecules such as P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule (VCAM) [81]. Recent evidence suggests that exosomes (i.e. small microvesicles with cytosolic contents enclosed within a lipid bilayer) are generated by perivascular macrophages and can influence endothelial behaviour [84, 85]. Exosomes encase RNA species from donor macrophages including microRNAs [83] and can fuse with the cellular membrane of adjacent endothelial cells and deliver their contents directly into target cells. MicroRNAs are short sequences of RNA (20–25 nucleotides in length) that bind to the untranslated tails of specific complementary messenger RNA (mRNA) sequences and limit further translation of or degrade peptide sequences from the mRNA template. These microRNAs have the capacity to simultaneously inhibit the peptide synthesis of multiple mRNA targets. One microRNA known as miR-150 has been successfully delivered from exosomes to endothelial monolayers in vitro and found to alter endothelial cell behaviour by 284

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increasing cell migration [81, 86]. It is tempting to speculate that macrophage-derived exosomes are generated in systemic inflammatory states and disrupt endothelial barrier function during sepsis, but this has not yet been confirmed within the microcirculation and perivascular space in patients with sepsis [81]. Macrophages appear to have the capacity to directly influence endothelial cell function and structure by direct cell-to-cell contact with endothelial cells. Macrophages express the angiopoietin receptor, which is a tyrosine kinase known as Tie2 (discussed in detail below). Interaction between angiopoietin (Ang)-2 and Tie2 promotes endothelial growth, cell survival and angiogenesis. This direct cell contact between macrophages and endothelial cells might be important in tissue repair and for reestablishing endothelial barrier function during the recovery phase of sepsis [81, 82]. Reciprocal interactions between endothelial cells and macrophages maintain tissue macrophage viability and promote a microenvironment for macrophage differentiation and maturation [81, 87] Endothelial cells promote differentiation to M2-like macrophages, which have an anti-inflammatory phenotype and rely on oxidative metabolism to support resolution of tissue injury and repair.

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Endothelial cell MPs MPs are vesicles that range in size from 0.1 to 2 lm and are shed from the plasma membrane of multiple cell types upon activation or apoptosis [88, 89]. MPs contain many biologically active molecules, including proteins, lipids, mRNAs and microRNAs. The cellular source of circulating MPs is revealed by the antigens they express, which allows investigation of the function of cell-specific MPs. It was shown that MPs are present at low levels in the circulation of healthy subjects and originate predominantly from platelets [90]. Proinflammatory cytokines and bacterial products can induce shedding of MPs from endothelial cells that contain ultra-large von Willebrand factor multimers, which potently promote the formation of platelet aggregates and increase their stability [91]. Activated monocytes release MPs expressing TF [92]. Patients with septic shock had elevated total MP levels regardless of the presence of DIC, whereas levels of endothelium- and leucocytederived MPs were positively correlated with DIC status [93]. Of note, MPs can also exert anticoagulant effects. Anionic phospholipids exposed by MPs can promote the assembly of anticoagulant proteins such as TFPI, thrombomodulin, EPCR and protein S. APC can stimulate the release of MPs from endothelial cells, which facilitates the efficient inactivation of factors Va and VIIIa by MP-derived EPCR. Several cytoprotective properties associated with APC could be reproduced by APC-positive MPs in vitro [94]. Additionally, recombinant human APC, which until recently was registered for the treatment of severe sepsis, has been found to induce an increase in circulating APC-positive MPs. This suggests a functional role of anticoagulant and cytoprotective MPs in the in vivo effects of APC [95]. The endothelium and the microcirculation The endothelium plays a pivotal role in the normal function of the microcirculation. Sepsis is associated with a decreased flow velocity in the microcirculation and a reduced density of perfused capillaries [96]. Alterations in the microcirculation in sepsis are characterized in particular by heterogeneous perfusion of tissues due to a lack, or intermittent perfusion, of capillaries adjacent to those with normal perfusion [97]. Heterogeneous perfusion of the microvascular circulation disturbs tissue oxygenation and leads to hypoxic areas even in the presence of preserved total blood flow to an

Review: Endothelial dysfunction in sepsis

organ [98]. Microvascular dysfunction has been linked to organ failure and mortality in patients with sepsis [96]; disturbed function of the vascular endothelium plays a pivotal role. The glycocalyx is a thin layer of glucosaminoglycans that covers the surface of the vascular endothelium [43]. The glycocalyx contains several components that are essential for normal homeostasis, including antithrombin and superoxide dismutase. One function of the glycocalyx is to reduce adhesion of leucocytes and platelets to endothelial cells. Sepsis results in the degradation of the glycocalyx, leading to enhanced adhesion of inflammatory cells and injury [44]. Other pathological responses implicated in microvascular dysfunction include activation of coagulation (resulting in thrombosis in the microvasculature), alterations in red cell deformability and impaired release of nitric oxide [99, 100]. Although these responses that contribute to microvascular dysfunction are clearly detrimental in the context of severe sepsis, they are important for host defence against invasive infection by limiting dissemination of the infection and recruiting cells to the infectious site. Of note, it has been reported that microcirculatory function is improved by several anticoagulants, including APC, antithrombin and low molecular weight heparin [101–104]. It is interesting that a modified antithrombin that is unable to bind to the vascular endothelium but with intact anticoagulant properties failed to improve the microcirculation in animals with sepsis, suggesting that the anticoagulant effect per se is not sufficient to improve microcirculatory function [103]. Endothelial barrier function Endothelial barrier dysfunction and microvascular leak critically contribute to the pathogenesis of organ failure in sepsis and of sepsis-related complications such as acute lung injury [105]. The vascular barrier consists of endothelial cells, together with cell–cell junctions and extracellular components such as the glycocalyx. Cell–cell junctions include adherens junctions, mainly composed of vascular endothelial (VE)-cadherin, and tight junctions (the zona occludens), predominantly consisting of occludins and claudins [106]. Under normal conditions, the endothelial barrier is semi-permeable, allowing transport of fluids and solutes from blood to tissues. In sepsis, however, barrier function is disturbed, resulting in enhanced passage of proteins and solutes outside ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2015, 277; 277–293

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Animation 1 This animation describes in a time lag fashion the pathophysiology behind endothelial barrier dysfunction in septic shock. Watch the animation here. Please note that the version of the video with narration will be up shortly.

the circulation, and oedema (Animation 1). Several mediators are involved in maintaining vascular barrier function (see Table 1).

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also transactivate S1P1 signalling to promote endothelial barrier function [109]. For example, PAR1 can influence vascular stability and function via S1P1 signalling. APC potently inhibits thrombin-induced vascular hyperpermeability by a mechanism dependent on transactivation of S1P1 [110], whereas thrombin-induced vascular hyperpermeability is dependent on another S1P receptor, S1P3 [111]. Notably, activation of PAR1 by low doses of thrombin can (like APC) lead to a barrier protective effect [112] and thrombin can transactivate PAR1–PAR2 heterodimers in late sepsis, which is also barrier protective [72]. Recently, a key role for S1P2 in the permeability and inflammatory responses of the vascular endothelium during endotoxaemia was revealed [113]. Downstream signalling of S1P2 in vascular inflammation included the activation of the stress-activated protein kinase and nuclear factor kappa light-chain enhancer of B-cell (NF-jB) pathways. Several S1P analogues have been developed, with the aim of preserving vascular integrity, for possible clinical use [109].

Vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is a glycoprotein produced by endothelial and lung epithelial cells, as well as by leucocytes and platelets [106]. VEGF regulates vascular permeability through binding to VEGF receptors 1–3, which are endothelial-cell-specific membrane tyrosine kinase receptors [107]. Activation of VEGF receptor 2 dissociates VE-cadherin from the adherens junction resulting in increased vascular permeability [108]. Although the results of preclinical studies to investigate the role of VEGF in sepsis have been inconsistent [106], a pilot study to test an antiVEGF antibody (bevacizumab) in patients with septic shock was announced in 2010 (clinicaltrials.gov identifier NCT01063010); the status of this trial is currently unknown. Sphingosine-1-phosphate Sphingosine-1-phosphate (S1P) is an endogenous bioactive sphingolipid produced in many types of cells that are highly abundant in plasma and regulate endothelial barrier function by the activation of its G-protein-coupled receptor S1P1 [109]. The interaction between S1P and S1P1 on endothelial cells enhances (i) vascular barrier function by downstream activation of small GTPases, (ii) cytoskeletal reorganization, (iii) adherens junction and tight junction assembly and (iv) focal adhesion formation. Other barrier-enhancing agents may 286

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Fibrinopeptide Bb15–42 Fibrinopeptide Bb15–42 is a cleavage product of fibrin that binds to VE-cadherin and stabilizes interendothelial junctions [114]. Administration of fibrinopeptide Bb15–42 may be of therapeutic value in sepsis. Treatment with this peptide preserved endothelial barrier function in two different shock models, induced by Dengue virus and LPS, by inhibiting stress-induced opening of endothelial cell adherens junctions; fibrinopeptide Bb15–42treated animals showed improved survival rates and reduced haemoconcentration and fibrinogen consumption [115]. In a pig model of haemorrhagic shock, fibrinopeptide Bb15–42 improved pulmonary and circulatory function and reduced plasma IL-6 levels and neutrophil influx into the myocardium, liver and small intestine [116]. Moreover, in a murine polymicrobial sepsis model, fibrinopeptide Bb15–42 treatment reduced proinflammatory cytokine levels in the lung, liver and blood, decreased neutrophil infiltration into the lung and attenuated liver damage, possibly through maintaining vascular integrity and suppressing vascular leakage [117]. In several models of acute lung injury, produced by airway exposure to LPS or acid, fibrinopeptide Bb15–42 reduced proinflammatory cytokine levels, neutrophil influx and vascular leak; moreover, fibrinopeptide Bb15–42 enhanced bacterial clearance and survival in mice with acid aspiration-induced lung injury subsequently

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challenged with Pseudomonas aeruginosa [118]. Because of its myocardial protective effects in experimental ischaemia–reperfusion injury [114], fibrinopeptide Bb15–42 (FX06) has been evaluated in a multicentre Phase IIa clinical trial in patients with myocardial infarction; in this study, fibrinopeptide Bb15–42 significantly reduced the size of the necrotic core zone of infarcts [119]. As such, fibrinopeptide Bb15–42 is a promising therapeutic agent, although its mechanism of action remains unclear. Robo4 and Slit Endothelial cells express the receptor Robo4 which, after binding to its ligand Slit, inhibits inflammation-induced endothelial permeability by consolidation of adherens junctions and modification of cytoskeletal dynamics [41, 120, 121]. It is likely that Slit stabilizes the vasculature by enhancing VE-cadherin localization to the cell surface. Indeed, it was shown that Slit2N, an active fragment of Slit2, increased VE-cadherin and p120-catenin localization to the endothelial cell surface [41]. Accordingly, Slit2N reduced endothelial cell monolayer permeability in vitro caused by inflammatory mediators, including VEGF, LPS, TNF-a and IL-1b [41, 121]. In vivo, Slit2N attenuated the accumulation of neutrophils and protein exudates in the alveolar space of LPS-treated mice. This effect of Slit2N was absent in Robo4-deficient mice, demonstrating the importance of this receptor and also suggesting that the effect of Slit2N is endothelial cell specific as Robo4 expression was restricted to this cell type [121]. In addition, the Slit2N-dependent reduction in endothelial cell monolayer permeability was inhibited in the presence of a VE-cadherin blocking antibody, confirming the importance of the action of Slit2N on VE-cadherin in vivo. Similarly, in mice with abdominal sepsis caused by caecal ligation and puncture, Slit2N inhibited the leakage of Evans Blue dye in the kidney and spleen, indicating preserved vascular barrier function and improved survival. Slit2N also inhibited lung permeability in a mouse model of avian H5N1 influenza [41]. Hence, the Slit–Robo4 interaction is important for maintaining vascular integrity in multiple settings, and Slit2N may be an attractive therapeutic agent in conditions associated with disruption of the endothelial barrier, including sepsis. Recently, a novel pathway important for endothelial cell integrity that functions independently from NF-jB has been identified [122]. Specifically, IL-1b

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was shown to induce endothelial permeability via activation of the adaptor MyD88 by a route that did not rely on NF-jB activation. Rather, MyD88, which also functions as the common adaptor of Toll-like receptor (TLR) signalling, activated a distinct NF-jB-independent pathway, through the small GTPase ADP-ribosylation factor 6 (ARF)6 and its activator ARF nucleotide-binding site opener (ARNO; also known as CYTH2). ARNO binds directly to MyD88, suggesting that MyD88–ARNO– ARF6 functions as a proximal IL-1b and TLR signalling pathway distinct from that mediated by NF-jB. SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhanced vascular stability and improved outcomes in animal models of sterile inflammation [122]. The relevance of MyD88–ARNO–ARF6 signalling and the effect of SecinH3 have not yet been investigated in human sepsis. Ang-1 and Ang-2 Ang-1 and Ang-2 are widely studied biomarkers of endothelial activation and dysfunction in sepsis [123]. Ang-1 is produced constitutively, in particular by pericytes and smooth muscle cells, whereas Ang-2 is produced by endothelial cells where it is stored in Weibel–Palade bodies for quick release upon exposure to inflammatory stimuli. Ang-1 and Ang-2 bind to and inhibit the endothelial cell Tie2 receptor. Under physiological conditions, circulating Ang-1 levels exceed those of Ang-2, enabling preferential interaction between Ang-1 and the Tie2 receptor. This triggers pro-survival pathways and inhibits pro-inflammatory responses, resulting in endothelial cell quiescence. The inflammatory response that accompanies sepsis causes exocytosis of Weibel–Palade bodies and release of Ang-2, which results in a shift towards Ang-2–Tie2 interaction. This interaction promotes pro-inflammatory and pro-thrombotic pathways, microvascular leak and angiogenic stimuli. In clinical sepsis, a rise in the Ang-2:Ang-1 ratio, normally low under nonstressed conditions, is an indicator of acute vascular dysfunction [106]. In accordance with this, elevated Ang-2 levels were found to be correlated with severity of illness and adverse outcomes in patients with sepsis [124, 125]. The functional role of Ang-2 was illustrated by experiments with heterozygous mice missing one Ang-2 allele; these animals were partially protected against vascular leak, acute lung injury and death in models of caecal ligation and ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2015, 277; 277–293

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puncture and LPS-induced toxicity [126]. Ang-1– Tie2-targeted strategies have been tested in preclinical sepsis models. The synthetic Tie2 agonist

vasculotide protected against vascular leak and mortality in polymicrobial sepsis in mice [127]. Similarly, vasculotide and the Ang-1-mimetic

Table 2 Potential new treatment options to prevent or treat endothelial barrier dysfunction in sepsis Therapeutic agent

Proposed mechanism of action

Current developmental status

Selepressin and other

Potent vasopressors that specifically

Phase II clinical trials [138]

V1A receptor agonists

bind to V1A receptors reducing permeability and limiting oedema formation

C’5a monoclonal antibody

Blocks C’5a anaphylotoxin from binding to its receptor

Early clinical trials planned for sepsis [139]

and prevents excess endothelial permeability and C5a-induced apoptosis Recombinant human

Promote APC formation which

Available in Japan, in Phase III

thrombomodulin and

prevents endothelial apoptosis;

clinical trials in Europe and North

APC variants

induce carboxypeptidase activity

and South America [140]

to degrade C’5a Ang-1/Tie2 agents

Inhibit NF-jB signalling and

Preclinical studies [127, 128]

possess endothelial cell anti-apoptotic activity S1P1 agonists

Stimulate VE-cadherins,

Preclinical studies [109]

actin polymerization at adherens junctions of the endothelium Fibrinopeptide Bb15–42 (FXO6)

Slit2N agonists

Fibrin cleavage product that

Phase II clinical trials in myocardial

binds VE-cadherin and

infarction, sepsis preclinical and

stabilizes interendothelial junctions

early clinical studies [118, 119]

Stabilize tight junctions between

Preclinical studies [41]

endothelial cells Pepducins

Lipidated peptides that are super-agonists

Preclinical studies [136]

of PAR2, inducing Rac-mediated barrier stabilization Anti-HMGB1 mAb

Blocks HMGB1-mediated loss of

Preclinical studies [131–135]

endothelial barrier function and upregulation of cytokine generation and of adhesion molecules Anti-VEGF receptor 2 mAb

Blocks VEGF2-mediated breakdown

Early clinical trials [108]

of VE-cadherin from the adherens junction in endothelial cells Ang, angiopoietin; S1P, sphingosine 1 phosphate receptor; V1A, vasopressin 1A receptor; C’, complement; APC, activated protein C; NF-jB, nuclear factor kappa light-chain enhancer of B cells; VE, vascular endothelium; Tie2, tyrosine kinase with immunoglobulin-like and epithelial growth factor-like domains; PAR, protease-activated receptor; Rac, a subfamily of GTPases; HMGB1, high-mobility group box 1; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody. 288

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MAT.Ang-1 attenuated vascular leak induced by LPS in mice [128, 129]. Strategies that either enhance the Ang-1–Tie2 interaction or inhibit the effects of Ang-2 warrant further investigation in sepsis. High-mobility group box 1 High-mobility group box 1 (HMGB1), first described as a DNA-binding nuclear protein in eukaryotic cells, regulates chromosomal replication, transcription and DNA repair [130]. It has subsequently been found that HMGB1 has a critical role in the innate immune response to infection and injury as a late-acting, proinflammatory cytokine-like protein. HMGB1 release from either activated or necrotic cells (particularly myeloid and endothelial cells), but not from apoptotic cells, results in this inflammatory action [131, 132]. The endothelial lining is both a source of HMGB1 release in sepsis and a target of HMGB1-mediated inflammatory actions during septic shock or other severe forms of noninfectious tissue injury. HMGB1 functions as an ‘alarmin’, or damageassociated molecular pattern, inducing and maintaining the acute inflammatory response. Elevated circulating levels of HMGB1 are detected by a series of receptors expressed within the microcirculation including the receptor for advanced glycated end products, TLR2 and TLR4 [130, 131]. HMGB1 disrupts endothelial barriers, alters the actin filament cytoskeleton, impairs tight junctions, promotes the release of large quantities of IL1a as well as an array of other cytokines and chemokines, and stimulates enhanced expression of cell surface adhesion components such as ICAM1 and VCAM1 on endothelial membranes [133–135]. Antibodies directed against HMGB1 can reverse many of these changes seen in experimental animal models of sepsis, indicating that clinical development of HMGB1 inhibitors should be considered (see Fig. 3 and Table 2). Conclusion Recovery and maintenance of the endothelial barrier is critical to survival in sepsis. New therapeutic targets for the management of sepsis at the level of the endothelium might become clinically applicable as the cellular and molecular mechanisms that cause barrier dysfunction become better understood. Agents are in development that promote cell survival by targeting mitochondria and cellular energetics [7, 35, 36]. Recombinant variants of APC, which have been specifically engineered to

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remove their anticoagulant properties and promote their cytoprotective capacity, are possible novel treatments for sepsis [110]. Agents are also in development that directly protect the endothelial barrier, including Ang-1 and Tie2 agonists [127–129], S1P1 agonists [109], fibrinopeptide Bb15–42 therapy [118, 119] and Slit2N agonists, by stabilizing tight junctions between endothelial cells [121]. Another strategy is the use of pepducins, small lipidated peptides that can readily cross cell membranes and either block PAR1/RhoA barrier disruption or function as super-agonists of PAR2/Rac-mediated barrier stabilization, as potential therapies for septic shock [136, 137]. Table 2 provides a summary of the novel treatment options now in clinical development that specifically target endothelial barrier function in sepsis [138–140]. Novel interventions directed at re-establishing endothelial barrier function in sepsis could be a major addition to the therapeutic armamentarium at a time when antibiotics are failing and results with other adjuvant therapies have been disappointing. This is an exciting area of research at present, and clinical trials should soon provide information to determine whether targeting the endothelium will benefit patients in septic shock. Conflict of interest statement Our institution receives a grant from Asahi Kasei to run the clinical coordinating centre for soluble thrombomodulin.

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Correspondence: Steven M. Opal MD, Chief, Infectious Disease Division, Memorial Hospital of Rhode Island, 111 Brewster Street, Pawtucket, RI 02860, USA. (fax: 401-729-2795; e-mail: [email protected]).

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