Marta Palomo

Endothelial Dysfunction after Hematopoietic Stem Cell Transplantation: Role of the Conditioning Regimen and the Type of Transplantation

There is endothelial activation and damage in hematopoietic stem cell transplantation (HSCT). The impact of the conditioning and type of HSCT on endothelial dysfunction in the early phases of HSCT has been evaluated. Plasma samples were obtained before and at different times after autologous and allogeneic HSCT with and without early complications. Changes in soluble markers of endothelial damage (VWF, ADAMTS-13, sVCAM-1, sICAM-1, and sTNFRI) were measured. There were changes in all markers evaluated that followed different patterns in auto and allo settings. For VWF and sTNRI, progressive increases from day Pre to day 14 and to day 21 were observed in the auto and the allo group, respectively. ADAMTS-13 activity correlated inversely with VWF levels. Levels of sVCAM-1 decreased until day 7, and raised significantly to day 14 and to day 21 in the auto and the allo HSCT, respectively. No significant changes were detected for sICAM-1. Our results confirm that there is endothelial damage at the early phases of HSCT, apparently induced by the consecutive effects of the conditioning, the proinflammatory agents used during transplantation, the translocation of endotoxins across the damaged gastrointestinal tract, and the engraftment. However, the comparative analysis between patients with and without complications suggests that none of these markers has diagnostic or prognostic value.

Defibrotide Prevents the Activation of Macrovascular and Microvascular Endothelia Caused by Soluble Factors Released to Blood by Autologous Hematopoietic Stem Cell Transplantation

Endothelial activation and damage occur in association with autologous hematopoietic stem cell transplantation (HSCT). Several of the early complications associated with HSCT seem to have a microvascular location. Through the present study, we have characterized the activation and damage of endothelial cells of both macro (HUVEC) and microvascular (HMEC) origin, occurring early after autologous HSCT, and the potential protective effect of defibrotide (DF). Sera samples from patients were collected before conditioning (Pre), at the time of transplantation (day 0), and at days 7, 14, and 21 after autologous HSCT. Changes in the expression of endothelial cell receptors at the surface, presence and reactivity of extracellular adhesive proteins, and the signaling pathways involved were analyzed. The expression of ICAM-1 at the cell surface increased progressively in both HUVEC and HMEC. However, a more prothrombotic profile was denoted for HMEC, in particular at the time of transplantation (day 0), reflecting the deleterious effect of the conditioning treatment on the endothelium, especially at a microvascular location. Interestingly, this observation correlated with a higher increase in the expression of both tissue factor and von Willebrand factor on the extracellular matrix, together with activation of intracellular p38 MAPK and Akt. Previous exposure and continuous incubation of cells with DF prevented the signs of activation and damage induced by the autologous sera. These observations corroborate that conditioning treatment in autologous HSCT induces a proinflammatory and a prothrombotic phenotype, especially at a microvascular location, and indicate that DF has protective antiinflammatory and antithrombotic effects in this setting.

The Release of Soluble Factors Contributing to Endothelial Activation and Damage after Hematopoietic Stem Cell Transplantation Is Not Limited to the Allogeneic Setting and Involves Several Pathogenic Mechanisms

This study evaluated the relative impact of the intensity of the conditioning regimen and the alloreactivity in the endothelial dysfunction occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It involved a comparative analysis of the effect of incubating human umbilical vein endothelial cells (ECs) with serum samples from patients receiving autologous HSCT (auto-HSCT) or unrelated donor allo-HSCT. In both groups, blood samples were collected through a central line before conditioning (Pre), before transplantation (day 0), and at days 7, 14, and 21 after transplantation. Changes in the expression of EC receptors and adhesion proteins, adhesion of leukocytes and platelets under flow, and signaling pathways were analyzed. Endothelial activation and damage were observed in both groups, but with differing patterns. All markers of endothelial dysfunction demonstrated a progressive increase from day Pre to day 14 in the auto-HSCT group and exhibited 2 peaks of maximal expression (at days 0 and 21) in the allo-HSCT group. Both treatments induced a proinflammatory state (ie, expression of adhesion receptors, leukocyte adhesion, and p38 MAPK activation) and cell proliferation (ie, morphology and activation of ErK42/44). Prothrombotic changes (ie, von Willebrand factor expression and platelet adhesion) predominated after allo-HSCT, and a proapoptotic tendency (ie, activation of SAPK/JNK) was seen only in this group. These findings indicate that endothelial activation and damage after HSCT also occur in the autologous setting and affect macrovascular ECs. After the initial damage induced by the conditioning regimen, other factors, such as granulocyte colony-stimulating factor (G-CSF) toxicity, engraftment, and alloreactivity, may contribute to the endothelial damage seen during HSCT. Further studies are needed to explore the association between this endothelial damage and the vascular complications associated with HSCT.

Distinct Deleterious Effects of Cyclosporine and Tacrolimus and Combined Tacrolimus–Sirolimus on Endothelial Cells: Protective Effect of Defibrotide

Endothelial dysfunction seems to be a key factor in the development of several complications observed early after hematopoietic stem cell transplantation (HSCT). The conditioning regimen and many other factors associated with the procedure are responsible for this endothelial damage. The effects of immunosuppressive agents on endothelial function have not been explored in detail. We evaluated the effects of 3 drugs commonly used in HSCT: 2 calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus (TAC), and an inhibitor of mTOR, sirolimus (SIR). We also evaluated the effect of the combination of TAC and SIR (TAC+SIR), which is used increasingly in clinical practice. Microvascular endothelial cells (HMEC-1) were exposed to these drugs to evaluate changes in (1) intercellular adhesion molecule (ICAM)-1 expression on the cell surface, assessed by immunofluorescence labeling and expressed as the mean gray value (MGV); (2) reactivity of the extracellular matrix (ECM) toward platelets, upon exposure of the ECM to circulating blood; and (3) whole-blood clot formation, assessed by thromboelastometry. Studies were conducted in the absence and presence of defibrotide (DF) to assess its possible protective effect. The exposure of HMEC-1 to CSA and TAC+SIR significantly increased the expression of ICAM-1 (157.5 ± 11.6 and 153.4 ± 9.5 MGV, respectively, versus 105.7 ± 6.5 MGV in controls [both P < .05]). TAC applied alone increased ICAM-1 slightly (120.3 ± 8.2 MGV), and SIR had no effect (108.9 ± 7.4 MGV). ECM reactivity increased significantly only in response to CSA (surface covered by platelets of 41.2% ± 5.4% versus 30.1% ± 2.0%, P < .05). DF attenuated all these changes. No significant changes in the viscoelastic properties of clot formation were observed in any condition with blood samples incubated in vitro. In conclusion, CSA and TAC+SIR had a proinflammatory effect, but only CSA exhibited an additional prothrombotic effect. Interestingly, DF exerted clear protective anti-inflammatory and antithrombotic effects on the endothelium.

NFκB in the Development of Endothelial Activation and Damage in Uremia: An In Vitro Approach

Impaired hemostasis coexists with accelerated atherosclerosis in patients with chronic kidney disease (CKD). The elevated frequency of atherothrombotic events has been associated with endothelial dysfunction. The relative contribution of the uremic state and the impact of the renal replacement therapies have been often disregarded. Plasma markers of endothelial activation and damage were evaluated in three groups of patients with CKD: under conservative treatment (predialysis), on hemodialysis, and on peritoneal dialysis. Activation of p38 MAPK and the transcription factor NFκB was assessed in endothelial cell (EC) cultures exposed to pooled sera from each group of patients. Most of the markers evaluated (VCAM-1, ICAM-1, VWF, circulating endothelial cells) were significantly higher in CDK patients than in controls, being significantly more increased in the group of peritoneal dialysis patients. These results correlated with the activation of both p38 MAPK and NFκB in EC cells exposed to the same sera samples, and also to the peritoneal dialysis fluids. Hemodialysis did not further contribute to the endothelial damage induced by the uremic state observed in predialysis patients, probably due to the improved biocompatibility of the hemodialysis technique in recent years, resulting in lower cellular activation. However, peritoneal dialysis seemed to exert a significant proinflammatory effect on the endothelium that could be related to the high glucose concentrations and glucose degradation products present in the dialysis fluid. Although peritoneal dialysis has been traditionally considered a more physiological technique, our results raise some doubts with respect to inflammation and EC damage.

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations.

Quantitative and qualitative analysis of proteins in fresh frozen plasma obtained from whole blood donations and prepared with two photochemical treatments

Thirty-six fresh frozen plasma (FFP) units obtained from whole blood donations were used for 12 replicate experiments. For each replicate experiment, three ABO-matched FFP units were pooled and divided into three units containing different volumes of identical plasma. One unit was used as control FFP, one unit was treated with methylene blue plus visible light and one unit was treated with amotosalen and UVA light. The overall coagulation factor levels were better maintained in untreated FFP than in photochemically treated plasma. However, treated-plasma by both photochemical methods maintained coagulation factor levels that met or exceeded the European Pharmacopeia requirements for therapeutic plasma.

Endothelin-1 levels predict endothelial progenitor cell mobilization after acute myocardial infarction.

INTRODUCTION Endothelin-1 (ET-1), circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) are well-known modulators of endothelial function with important cardiac effects after an acute myocardial infarction. However, the relationship between them has never been assessed. The objective of the present study was to establish the relationship between ET-1, CEC, and EPC concentrations after ST-elevation myocardial infarction (STEMI). METHODS Endothelin-1, CEC, and EPC levels were measured in 61 patients presenting with a first STEMI. Samples were withdrawn acutely 6-24h and 1week after admission. Assessments included reperfusion outcomes (angiography), left ventricular ejection fraction (echocardiography), and 30-day mortality. RESULTS Mean age was 60.6±12.6years and 45 (74%) were males. Higher ET-1 plasma levels were associated with lower EPC count after 1week (7.45±2.53pg/ml if EPCs in the first quartile vs 5.72±1.49pg/ml if EPCs in the fourth quartile; P=0.04). In contrast with CEC and EPC count, higher ET-1 concentrations on admission were associated with Killip≥2 (9.92±2.01pg/ml vs 7.32±2.13pg/ml; P<0.001), post-reperfusion thrombolysis in myocardial infarction (TIMI) <3 (8.65±2.86pg/ml vs 5.87±1.93pg/ml; P=0.002), myocardial blush grade (MBG) <3 (7.46±2.48pg/ml vs 5.99±2.01pg/ml; P=0.004) and higher 30-day mortality (10.29±2.02pg/ml vs 6.57±2.20pg/ml; P=0.005). CONCLUSIONS In STEMI patients, high ET-1 levels on admission predict a lower EPC mobilization after 1week. Endothelin-1 provides better clinical, angiographic and echocardiographic information for prognosis than do CEC and EPC concentrations.

Effect of Two Different Dialysis Membranes on Leukocyte Adhesion and Aggregation

Aims: We evaluated modifications in formation of heterotypic platelet-leukocyte aggregation induced by dialysis through cellulosic or synthetic membranes and evaluated the effects of such procedures promoting adhesive interactions between leukocytes and normal endothelial cells (ECs). Methods: Samples were obtained from arterial and venous lines at baseline, after 15 and 120 min of hemodialysis. Heterotypic aggregation was assessed using flow-cytometric techniques. Experiments to determine leukocyte adhesion to ECs were performed in parallel plate perfusion chambers at 450 s–1. Results: Patients dialyzed with a cellulosic membrane showed a significantly higher baseline granulocyte heterotypic aggregation (median 22.5%, range 8.6–32%) versus healthy subjects (median 10%, range 3.2–14.6%; p < 0.05). Granulocyte heterotypic aggregation values remained increased throughout the hemodialysis session not only in the arterial line (median 18 and 24.5%, range 7–30 and 8.7–36% at 15 and 2 h, respectively) but also in the venous line (median 20 and 25%, range 8.6–32 and 11.5–35% at 15 min and 2 h, respectively). Basal lymphocytes heterotypic aggregation values observed in uremic patients were 6% (0.1–7.1%) versus 1.0% (0.5–2.8%) in the control group (p < 0.05). The increase remained during the hemodialysis session both in the arterial line (median 5 and 4%, range 0.2–14 and 0.5–7.1 % at 15 min and 2 h, respectively) and in the venous line (median 7 and 7%, range 1.4–14 and 0.5–10.6% at 15 min and 2 h). In contrast, patients dialyzed with a synthetic membrane showed a decreased basal granulocyte heterotypic aggregation compared to healthy subjects (median 3.5 vs. 10%, range 2.8–7 vs. 3.2–14.6%, respectively; p < 0.05). For lymphocytes, basal heterotypic aggregation values were 0.2% (range 0.1–0.5%) in dialyzed patients vs. 0.98% (range 0.5–2.8%) in healthy subjects (p < 0.05), without changes throughout the dialysis session. Changes in leukocyte adhesion during hemodialysis did not reach statistical significance with either hemodialysis membrane. Our studies confirm a differential activation of platelets and leukocytes depending on the nature of the dialysis membranes. However, activation of circulating cellular elements by hemodialysis procedures did not enhance cross-talk interactions between leukocytes and unaltered ECs.

Innovative strategies minimize engraftment syndrome in multiple myeloma patients with novel induction therapy following autologous hematopoietic stem cell transplantation

Autologous stem cell transplantation (PBSCT) is standard for young patients in MM and its TRM has decreased after the 2000s. Bortezomib and immunomodulatory agents (IMiDs) in MM have improved the outcome. However, they seem to boost pro-inflammatory stage increasing the incidence of engraftment syndrome (ES). Favorable factors in PBSCT such as G-CSF could increase inflammatory stage during transplant. Corticosteroids have shown an excellent response of ES and some authors propose them as prophylaxis for ES. The aim was to analyze the impact of G-CSF avoidance and corticosteroids’ prophylaxis in 170 patients diagnosed of MM treated with bortezomib/IMiDs that underwent PBSCT. We established three groups: Group-I [(G-CSF_administration), 60 patients (35%)], group-II [(nonG-CSF), 60 patients (35%)] and group-III [(nonG-CSF plus corticosteroid’s prophylaxis), 50 patients (30%)]. A decreased ES incidence among groups was observed: 62, 42, and 22% (P < 0.0001). The incidence of symptoms mimicking a capillary leak syndrome associated with ES dropped: 43, 32, and 0% (P = 0.03). The G-CSF avoidance and corticosteroids had impact over admission 24, 21, and 20 days (P = 0.001). The most important variables related to ES were HCT-CI >2 (p < 0.0001; HR 8.5) and risk groups (p < 0.0001; HR 7.2). Hence, G-CSF avoidance and corticosteroid’s prophylaxis decrease morbidity in patients undergoing PBSCT with MM treated with bortezomib/IMiDs.