The Role of the Endothelium in the Short-Term Complications of Hematopoietic SCT

REVIEW The role of the endothelium in the short-term complications of hematopoietic SCT

E Carreras1,3 and M Diaz-Ricart2

1Hematology Department, Hospital Clinic, Institut d’Investigacions Biome`diques Augustı´ Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; 2Hemotherapy-Hemostasis Department, Hospital Clinic, Institut d’Investigacions Biome`diques Augustı´ Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain and 3Spanish Bone Marrow Donor Program (REDMO), Josep Carreras International Foundation, Barcelona, Spain

In this review, we analyse the role of the endothelium Introduction in the development of several complications that appear soon after haematopoietic SCT (HSCT). Once it had been Haematopoietic SCT (HSCT) is a well-established ap- demonstrated that sinusoidal damage is the initiating proach for the treatment of several haematologic, meta- event of the sinusoidal obstruction syndrome, it was bolic and neoplastic disorders.1 However, HSCT is not an considered that other short-term complications with innocuous procedure and may be associated with several overlapping clinical manifestations, such as capillary leak early and late life-threatening complications. Those com- syndrome, engraftment syndrome, transplant-associated plications that appear early after HSCT are usually caused microangiopathy, diffuse alveolar haemorrhage and idio- by the toxicity of the conditioning regimen, infections or pathic pneumonia syndrome, could have an endothelial immune reactions (mainly GVHD). In addition, in this origin. During HSCT, endothelial cells (ECs) are initial phase, there is an interesting group of complications activated and damaged by several factors, including that do not have well-established origins. The most relevant conditioning, cytokines released by damaged tissues, are sinusoidal obstruction syndrome, capillary leak syn- endotoxins translocated through damaged mucosa, drugs drome, engraftment syndrome, transplant-associated mi- used in the procedure, the engraftment, and—in the croangiopathy (TAM), diffuse alveolar haemorrhage, and allogeneic setting—immunological reactions. The differ- idiopathic pneumonia syndrome. All these complications ent clinical syndromes that occur could be determined by share the following characteristics: they have an early onset the predominant phenotypic change in the ECs and the after HSCT, overlapping clinical manifestations, the location of this change (organ dependant or systemic). absence of well-defined clinical criteria for diagnosis (and Several translational studies have provided evidence of this consequently an unknown true incidence), the absence of endothelial dysfunction on the basis of analysis of soluble well-established treatments, and the tendency to evolve to markers, soluble forms of adhesion molecules, the an irreversible multiorgan dysfunction syndrome. enumeration of circulating ECs and microparticles, and morphologic and functional changes induced in cultured ECs. This increased knowledge has opened up a wide What is known about the pathogenesis of these syndromes? range of potential pharmacologic interventions to prevent or treat endothelial damage and, consequently, to improve Sinusoidal obstruction syndrome, commonly called hepatic the outcome of patients receiving HSCT. veno-occlusive disease (VOD), is the most frequent and well Bone Marrow Transplantation (2011) 46, 1495–1502; studied of these complications and the only one with well- doi:10.1038/bmt.2011.65; published online 4 April 2011 established clinical criteria for diagnosis.2,3 Its frequency Keywords: endothelial cells; endothelial damage; veno- ranges from 3% in autologous HSCT (auto-HSCT) to occlusive disease; haematopoietic SCT; microvasculature 50–60% in the allogeneic setting.2,4 In vivo studies conducted in rats that received monocrotaline show that the first morphologic change noted by EM is the loss of sinusoidal endothelial cell (EC) fenestration and the appearance of gaps in the sinusoidal EC barrier (Figure 1b). These studies also show that the sinusoidal ECs round up, and the RBC begin to penetrate into Correspondence: Dr E Carreras, Spanish Bone Marrow Donor Program the space of Disse beneath the ECs and dissect off the (REDMO), Josep Carreras International Foundation, Muntaner 383 endothelial lining (Figure 1c). Finally, the sloughed 2-2, 08021-Barcelona, Spain. E-mail: [email protected] sinusoidal lining cells embolize downstream and obstruct Received 31 January 2011; revised and accepted 17 February 2011; sinusoidal flow (Figure 1d). In these early stages, histolo- published online 4 April 2011 gical examinations show thickening of the subintimal Endothelial syndromes after HSCT E Carreras and M Diaz-Ricart 1496

Figure 1 Veno-oclusive disease pathogenesis. (a) Normal hepatic sinusoid; (b) Sinusoidal ECs damaged during conditioning round up favouring the appearance of gaps in the sinusoidal barrier; (c) RBC begin to penetrate into the space of Disse detaching the endothelial lining; (d) The sloughed sinusoidal lining cells embolize downstream and obstruct the sinusoidal ﬂow (sinusoidal obstruction syndrome). Images provided by Jordi Bozo.

zone that leads to the narrowing of the venular lumen and Table 1 Suspected pathogenesis of vascular endothelial syndromes an increased resistance to blood flow, which contribute after HSCT to the haemodynamic changes seen in this disease. Thus, Syndrome Supposed pathogenesis despite the possibility that other factors can contribute to VOD development, endothelial injury seems to be the VOD See text initiating event in the cascade of events leading to clinical CLS Diffusely injured capillary endothelium leads to the 5–7 leakage of intravascular fluids into interstitial space manifestation of this complication. ES Massive release of pro-inflammatory cytokines when new These observations of VOD, the fact that the pathogen- neutrophils cross the endothelial barrier when moving esis of the remaining early complication seems to have a from marrow niches to blood flow close relationship with the microvascular tree (Table 1), DAH Lung capillary endothelium damaged by conditioning and their overlapping clinical manifestations (Table 2), are plus engrafted neutrophils and silent infections, among other factors, allowing the leakage of RBC into the the main reasons these syndromes have been grouped under pulmonary alveoli the denomination of vascular syndromes or vascular TAM Generalized endothelial dysfunction with intravascular endothelial syndromes after HSCT. platelet activation and formation of platelet-rich thrombi within the microcirculation IPS Clinical and experimental IPS is associated with vascular EC injury in the lungs and increased permeability Why does HSCT lead to endothelial damage? produced by TNF, neutrophils and immune reactions

The endothelium is an active biologic interface between the Abbreviations: CLS ¼ capillary leak syndrome; DAH ¼ diffuse alveolar blood and all other tissues, and it mediates vaso-motor haemorrhage; EC ¼ endothelial cell; ES ¼ engraftment syndrome; tone, haemostatic balance and inﬂammatory reactions, IPS ¼ idiopathic pneumonia syndrome; TAM ¼ transplant-associated microangiopathy; TNF ¼ tumour necrosis factor; VOD ¼ veno-occlusive among other functions, throughout the circulatory system. disease. Several input stimuli can produce a local or systemic physiological endothelial activation. The term EC activation includes a wider spectrum of phenotypic changes in The proposed hypothesis is that, during HSCT, ECs the endothelium. This activation takes place in a graded can be activated and damaged by factors such as the manner rather than an ‘all or nothing’ response and differs chemoradiotherapy included in the conditioning regimen, according to several physiological variables, including the cytokines produced by the injured tissues, endogenous location of the vascular bed. When the activating stimulus microbial products translocated through damaged mucosal is too intense or persistent, it can produce a localized barriers,9 drugs used during the procedure (such as G-CSF or systemic endothelial dysfunction. Currently, the term or calcineurin inhibitors),10,11 and the complex process of dysfunction is broadly applied to states in which the EC engraftment. All these factors produce a physiological phenotype poses a net liability to the host.8 activation of the EC; however, if they are intense and

Bone Marrow Transplantation Endothelial syndromes after HSCT E Carreras and M Diaz-Ricart 1497 sustained, the EC activation can evolve into endothelial Moreover, there is clinical evidence suggesting that, damage. The different syndromes are determined by in the allogeneic setting, alloreactivity also has a role in the predominant phenotypic change (pro-inflammatory, the pathogenesis of these endothelial complications.12,13 pro-thrombotic, pro-apoptotic) and its localisation This could explain why these complications are more (systemic or organ dependant) (Figure 2). frequent in allogeneic rather than auto-HSCT, despite identical conditionings being used. In addition, they are more frequent among patients receiving HSCT from unrelated donors, especially if they are mismatched, Table 2 Clinical manifestations of the vascular endothelial and the procedures reducing the allogenicity of the graft syndromes after HSCT (such as T-cell depletion) reduce their incidence.14 Symptoms and signs VOD CLS ES DAH IPS TAM Vascular ECs constitute a target tissue for blood-borne executors of the immune system, that is, the antibodies and Usually starting 0–7 7–10 11–15 11–19 18–23 25–120 T-lymphocytes. Transmigration of lymphocytes through on day: the endothelial monolayer involves intimate contact Fever || | | Jaundice | between the two types of cells facilitating EC and T-cell Hepatomegaly | activation. In addition, T-cell–EC contact occurs twice, Weight gain ||| firstly, when the donor stem cells are administered and Oedemas || secondly, when the newly generated T-lymphocytes enter Ascites || Lung infiltrates ||| | | into the blood stream. This double wave of activation Dyspnoea ||| | | is also suggested by some in vitro studies showing a biphasic Hypoxia ||| | | evolution of EC activation after allogeneic HSCT Diarrhoea | (allo-HSCT).15 ||| | Renal dysfunction Finally, several experimental models propose that Neurological || dysfunction vascular ECs are targets for alloreactive and allospecific Evolution to MODS ||| | | T-lymphocytes in acute and chronic GVHD.16 These Predominant in: allo auto auto allo allo allo observations have led to speculations that this early endothelial damage, together with the chronic vascular Abbreviations: allo ¼ allogeneic HSCT; auto ¼ autologous HSCT; CLS ¼ inflammation observed during chronic GVHD, can have a capillary leak syndrome; DAH ¼ diffuse alveolar haemorrhage; role in the development of the vascular complications ES ¼ engraftment syndrome; IPS ¼ idiopathic pneumonia syndrome; MODS ¼ multiorgan dysfunction syndrome; TAM ¼ transplant-associated observed later in allo-HSCT, especially when GVHD microangiopathy; VOD ¼ veno-occlusive disease. occurs.17

Multiorgan dysfunction syndrome

DAH CLS ES ES VOD IPS TAM TAM TAM

Organ dysfunction

Endothelial Endothelial phenotype represents a net liability to the host dysfunction (capillary flow obstruction, fibrin-related aggregates, (pathologic) platelet and leukocyte adhesion, endothelial apoptosis)

Endothelial Pro-coagulant Inflammatory Increased activation Vasoconstriction (physiologic) status response permeability

Conditioning G-CSF CNI LPS/infections Engraftment Allo-reactivity

Hematopoietic stem cell transplantation

Figure 2 Common pathogenesis of the vascular endothelial syndromes developed early after HSCT. CLS, capillary leak syndrome; CNI, calcineurin inhibitors; DAH, diffuse alveolar haemorrhage; ES, engraftment syndrome; IPS, idiopathic pneumonia syndrome; LPS, lipopolysaccharide; TAM, transplant-associated microangiopathy; VOD, veno-occlusive disease.

Bone Marrow Transplantation Endothelial syndromes after HSCT E Carreras and M Diaz-Ricart 1498 Translational evidence of endothelial dysfunction conditioning in all patients. At the time of engraftment after HSCT or at the onset of GVHD, VWF and TM levels were significantly increased with respect to the baseline, espe- EC injury can be evaluated through the analysis of several cially in patients with acute GVHD grades II–IV, suggest- coagulation factors, soluble markers or adhesion ing that ECs could be injured. Nu¨rnberg et al.22 analysed molecules, the enumeration of circulating ECs (CECs) levels of plasminogen activator inhibitor type 1 and TM and microparticles (Figure 3), and the evaluation of and demonstrated that the increases in both markers morphologic and functional changes induced in cultured correlated with the number of vascular complications ECs. (VOD, sepsis, capillary leak syndrome) and to a lesser degree with GVHD. Takatsura et al.23 did not observe any impact of TBI on VWF and TM levels. Matsuda et al.24 Soluble markers and adhesion molecules investigated the serum levels of sVCAM-1, sE-selectin and Von Willebrand factor (VWF), soluble thrombomodulin sICAM-1 in HSCT, excluding patients with severe infec- (TM), TNF-a, plasminogen activator inhibitor type 1 tions. They showed that all soluble adhesion molecules and soluble adhesion molecules (sE-selectin, sICAM-1, increased after HSCT (day þ 30) and that levels were sVCAM-1) are the markers that have been most commonly higher after allo- compared with auto-HSCT. sE-selectin evaluated. and sVCAM-1 specifically increased in patients with Richard et al.18 investigated serum levels of VWF, TM, extensive chronic GVHD, and sVCAM-1 increased in sICAM-1 and TNF-a on days À7, þ 7 and þ 21 in patients patients with TAM. Park et al.25 evidenced significantly receiving auto- or allo-HSCT. Before HSCT, levels of VWF reduced VWF-cleaving protease activity in patients with and TM were significantly increased compared with VOD, even before conditioning. Luzzato et al.26 also controls. VWF and sICAM, but not TM, were significantly demonstrated an increase in VWF, TM and sE-selectin increased on days þ 7 and þ 21. In patients with VOD, after HSCT in a paediatric population. Interestingly, when only sICAM increased. Catani et al.19 analysed TM and comparing BM, peripheral blood or cord blood HSCT, Rio P-selectin weekly from day 0 to day þ 28 and observed et al.27 showed that the increase in VWF and TM levels was normal levels in all patients, except for those developing absent after cord blood HSCT, suggesting that a tolerance severe VOD. Zeigler et al.20 observed that VWF and TM exists between cord blood stem cells and the vascular were increased in all patients with TAM after HSCT. Salat wall. Recently, Cutler et al.28 analysed four biomarkers of et al.21 evidenced that VWF and TM rose significantly after endothelial injury. They concluded that elevations of VWF,

Endothelial cells Input signals: Output changes: Vasomotor tone Cell interaction Soluble Permebility Blood flow adhesion Soluble Coagulation Hemostatic balance Temperature molecules selectins factors Inflammatory response Glucose pH Cell proliferation Oxygenation sICAM-1 sE-selectin sTM Apoptosis Soluble mediators (cytokines, sVCAM-1 sP-selectin sTF endothelins, VEGF...) Microparticles VWF

Circulating endothelial Adhesion cells molecules Selectins Metalloproteases VCAM-1 P-selectin ICAM-1 E-selectin ADAMTS-13 Prostaglandins Transduction TM CEC signals NO TLR p38MAPK NFκB SAPK/JNK EC ERK 42/44 Detachment Akt

VWF TM TF ECM

Figure 3 ECs activation and damage. Grey boxes indicate the input and output signals participating in endothelial activation. White boxes indicate most usual biomarkers that allow the evaluation of the endothelial function. ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; Akt, a-serine/threonine-protein kinase; CEC, circulating endothelial cell; EC, endothelial cell; ECM, extracellular matrix; ERK, extracellular signal-regulated kinases; ICAM, inter-cellular adhesion molecule; MAPK, mitogen-activated protein kinase; NO, nitric oxide; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; TF, tissue factor; TLR, toll-like receptors; TM, thrombomodulin; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VWF, Von Willebrand factor. Images provided by Jordi Bozo.

Bone Marrow Transplantation Endothelial syndromes after HSCT E Carreras and M Diaz-Ricart 1499 TM and sICAM-1 before and early after HSCT may be Additionally, the potential diagnostic and/or prognostic useful for predicting VOD in patients receiving sirolimus. utility of the soluble markers as indicators of endothelium- By attempting to generate a more detailed picture of the related complications in HSCT was explored. Preliminary endothelial damage after different types of HSCT, our data shows that levels of soluble biomarkers of endothelial group obtained samples from more than 100 consecutive dysfunction are higher in patients with VOD, engraftment HSCTs.15,29,30 Samples were drawn before transplant syndrome and GVHD. Further studies with a larger (day Pre), at the time of transplant (day 0), and at different number of patients are planned to improve statistical times after auto- and allo-HSCT (days þ 7, þ 14 and þ 21) confidence and determine the applicability of one or more in patients with or without early complications. Patients of these proteins as suitable diagnostic or prognostic with sepsis were excluded. VWF, ADAMTS-13 activity, markers of disease. However, all these biological markers sVCAM-1, sICAM-1 and sTNFRI were measured. seem to be nonspecific, seriously hampered by numerous Changes in the levels of most of the markers evaluated host factors, and do not allow endothelial dysfunction to be were observed, and these changes followed different predicted or quantified. patterns depending on the type of transplant. VWF and sTNFRI increased progressively from day Pre to day þ 14 for auto-HSCT and to day þ 21 for allo-HSCT. CECs and microparticles ADAMTS-13 activity was inversely correlated with VWF Recently, two further specific markers of endothelial levels. Levels of sVCAM-1 decreased until day þ 7, and dysfunction have been described, that is, microparticles increased up to day þ 14 for auto-HSCT and to day þ 21 and CECs.31,32 The former have been detected as follicles for allo-HSCT (Figure 4). In contrast, no significant shed from various types of activated cells, such as platelets, changes were detected for sICAM-1. These results con- monocytes, polymorphonuclear leukocytes and ECs. firmed previous evidence that endothelial damage occurs in Pihusch et al.33 demonstrated that levels of microparticles the early phases in parallel with the following: (1) the increased in patients with acute GVHD, but not in patients conditioning, (2) the administration of pro-inflammatory without this complication or during conditioning or sepsis. (G-CSF) or immunomodulatory agents, (3) the develop- CECs can be isolated and quantified using diverse methods ment of mucositis, and (4) the onset of donor leukocyte and seem to be an excellent marker of endothelial engraftment. Moreover, the degrees of change in biomarker dysfunction. Elevated levels of CECs reflect the extent of levels correlated with the intensity of the conditioning endothelial damage in a variety of disorders. Woywodt treatment and were more relevant in myeloablative et al.34,35 demonstrated for the first time in HSCT that compared with reduced-intensity conditioning. patients receiving total body radiation have an earlier peak

VCAM-1 Auto-BEAM Allo-Cy/TBI 2.5 2.5 * 2 * 2

1.5 1.5 Ex-vivo 1 * 1 * 0.5 0.5 Ratio respect pre-value Ratio respect pre-value 0 0 Day Pre 0 +7 +14 +21 DayPre 0 +7 +14 +21

100 * 100 * 2 2 80 80 *

60 60 In-vitro 40 40

20 20 Gold particles/um 0 Gold particles/um 0 Day Pre 0+7+14 +21 Day Pre 0 +7 +14 +21

Control Auto - day +14 Allo - day +21

Figure 4 Adhesion molecule VCAM-1. Changes in levels of soluble VCAM-1 (a, b) in auto-HSCT (a) and allo-HSCT (b), and expression of VCAM on the surface of ECs (c, d and micrographs) in auto-HSCT (c) or allo-HSCT (d). Changes on soluble VCAM1 are expressed as ratios with respect to the Pre values. Bar diagrams represent the density of gold labelling (pixels/mm2). All auto-HSCT were conditioned with BEAM and allo-HSCT with Cy/TBI. *Po0.05 versus Pre condition.

Bone Marrow Transplantation Endothelial syndromes after HSCT E Carreras and M Diaz-Ricart 1500 of CECs compared with patients only receiving chemother- associated with HSCT are bound to the microvasculature.30 apy. Patients receiving reduced-intensity conditioning For this reason, we comparatively studied the pro- have significantly lower CEC counts than those receiving inflammatory and pro-thrombotic phenotypes induced by standard conditioning.35 the auto-HSCT environment in the macro- and micro- Our initial studies included the enumeration of CECs. vascular locations by using the corresponding cell models. However, we encountered technical problems mainly In the autologous setting, activation and damage of both because of low leukocyte counts in the early stages after types of cells occurred with similar signs of inflammation, HSCT, which made interpretation of the results quite although with a more reactive ECM towards platelets difficult. in cells of microvascular origin. This higher reactivity was characterized by increased expression of VWF and tissue factor on the ECM. Because tissue factor is a major In vitro studies with micro- or macrovascular EC cultures activator of the extrinsic pathway of blood coagulation, the EC cultures offer a unique experimental model with which synthesis and release of this protein onto the ECM may to evaluate endothelial activation and damage, as well as to have important thrombotic consequences at the micro- test agents for preventing or correcting the endothelial vasculature. dysfunction. The Regensburg university group led these studies for many years. Initially, they demonstrated that ionizing radiations produced apoptosis of micro- and Therapeutic strategies to reduce endothelial dysfunction macrovascular ECs, an effect that increased with the associated with HSCT presence of the bacterial endotoxins, like lipopolysaccharide.36 In addition, using human umbilical-vein ECs, they Several protective agents have been used to prevent or treat demonstrated that allogenicity has a direct role in EC VOD after HSCT.39 In general, results have been dis- toxicity, enhancing lipopolysaccharide-mediated EC toxi- appointing and not always reproducible. Recently, results city.9 Using microvascular ECs, they further demonstrated with defibrotide as a treatment40–42 or prophylaxis43 for that ECs incubated with fludarabine show an increased VOD, and occasional evidence of its possible efficacy in lysis by allogeneic MHC-restricted cytotoxic T-lympho- other endothelial complications, such as TAM,44 have cytes.37 Finally, they evaluated microvascular endothelial increased interest in this agent. Interestingly, children damage in vitro after allo-HSCT. They observed an increase receiving prophylactic defibrotide in a randomized study43 in the apoptotic activity of the sera from patients at the had a lower incidence of VOD and a significantly lower time of engraftment and preceding episodes of acute and incidence of GVHD. This observation seems to corroborate chronic GVHD and TAM. Serum levels of VCAM-1 within the suspected relationship between endothelial damage the first 100 days also correlated with GVHD.38 and acute GVHD. Many publications describe the bene- Our group, in collaboration with the Regensburg group, ficial effects of defibrotide as a pro-fibrinolytic, anti- examined the effect of the different types of HSCT and thrombotic and thrombolytic, anti-ischemic, anti-shock, the conditioning regimens applied on the integrity of the anti-atherosclerotic, and anti-rejection drug.45 Eissner endothelium through a series of in vitro studies.15,30 We et al.37 demonstrated in vitro that defibrotide protected applied a laboratory-based macrovascular cell model to ECs from fludarabine toxicity. explore more deeply the effect of the soluble plasma factors Our later studies show that defibrotide prevents the released during HSCT on resting ECs. We confirmed our inflammatory reaction through inhibition of p38 mitogen- previous in vivo results reporting that the allo- and the auto- activated protein kinase and a-serine/threonine-protein HSCT resulted in EC activation. The observed effect was kinase activation and of the pro-thrombotic phenotype characterized by an increase in the expression of cell- by blocking changes in the composition of the ECM surface adhesion receptors (Figure 4), leukocyte adhesion generated by the sera of patients receiving HSCT. These following exposure to circulating blood, and the activation initial observations could be extended to other drugs, such of the p38 mitogen-activated protein kinase. Additionally, as oligotide46 or several antioxidant agents, which have plasma from patients in both settings induced cell the capability to modulate endothelial activation and proliferation, which was measured by activation of dysfunction. ERK42/44. We observed an increased adhesion of platelets In summary, despite more than 15 years of analysing the on the extra-cellular matrix (ECM) generated when role of the endothelium in several of the complications exposed to blood under flow conditions. This effect observed after HSCT, our knowledge remains very limited, correlated with enrichment of the ECM with VWF, but and it will be necessary to delve further into these studies. was only clearly significant in the allogeneic setting. However, we have in front of us an attractive scenario that Similarly, apoptosis, assessed by measuring activation enables us to predict a future in which we could prevent of the SAPK/JNK pathway, was only observed in the these complications and improve the outcome of patients group of allo-HSCT recipients. These observations justify receiving HSCT. why complications with an inflammatory profile, such as engraftment syndrome and capillary leak syndrome, predominate in auto-HSCT, whereas those with a thrombotic basis, such as VOD or TAM, predominate in allo-HSCT. Conflict of interest Despite our previous observations being generated in the macrovascular cell model, most of the early complications The authors declare no conflict of interest.

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