Safety and efficacy of G-CSF after allogeneic hematopoietic cell transplantation using post-transplant cyclophosphamide: clinical and in vitro examination of endothelial activation.

Since 2021 the use of G-CSF was implemented in allo-HCT with PTCY-based prophylaxis with the aim of shortening the aplastic phase and reducing infectious complications. This study investigates the effectiveness of this change in protocol performed at our institution. One-hundred forty-six adults undergoing allo-HCT with PTCY-based prophylaxis were included, and among them, 58 (40%) received G-CSF. The median of days to neutrophil engraftment was shorter in the G-CSF group (15 vs. 20 days, p < 0.001). Patients receiving G-CSF had a lower incidence of day +30 bacterial bloodstream infections (BSI) than the rest (20.7% vs. 47.7%, p < 0.001). GVHD, SOS, and TA-TMA incidences were comparable between groups, and using G-CSF did not impact on survival. Endothelial activation was investigated using EASIX and by the measurement of soluble biomarkers in cryopreserved plasma samples obtained on days 0, +7, +14 and +21 of 39 consecutive patients (10 received G-CSF) included in the study. EASIX, VWF:Ag, sVCAM-1, sTNFRI, ST2, REG3α, TM and NETs medians values were comparable in patients receiving G-CSF and those who did not. Compared with allo-HCT performed without G-CSF, the addition of G-CSF to PTCY-based allo-HCT accelerated neutrophil engraftment contributing on decreasing BSI incidence, and without inducing additional endothelial activation.

Endothelial injury and dysfunction with emerging immunotherapies in multiple myeloma, the impact of COVID-19, and endothelial protection with a focus on the evolving role of defibrotide.

Patients with multiple myeloma (MM) were among the groups impacted more severely by the COVID-19 pandemic, with higher rates of severe disease and COVID-19-related mortality. MM and COVID-19, plus post-acute sequelae of SARS-CoV-2 infection, are associated with endothelial dysfunction and injury, with overlapping inflammatory pathways and coagulopathies. Existing treatment options for MM, notably high-dose therapy with autologous stem cell transplantation and novel chimeric antigen receptor (CAR) T-cell therapies and bispecific T-cell engaging antibodies, are also associated with endothelial cell injury and mechanism-related toxicities. These pathologies include cytokine release syndrome (CRS) and neurotoxicity that may be exacerbated by underlying endotheliopathies. In the context of these overlapping risks, prophylaxis and treatment approaches mitigating the inflammatory and pro-coagulant effects of endothelial injury are important considerations for patient management, including cytokine receptor antagonists, thromboprophylaxis with low-molecular-weight heparin and direct oral anticoagulants, and direct endothelial protection with defibrotide in the appropriate clinical settings.

EASIX and cardiac adverse events after allogeneic hematopoietic cell transplantation.

This study investigates the interaction between endothelial activation, indirectly measured using EASIX, and the probability of presenting cardiac adverse events (CAE) during the first year after allo-HCT. The 437 consecutive adults undergoing PB allo-HCT from 2012 and 2021 were included. EASIX was retrospectively calculated before and during the first 6 months after allo-HCT and transformed to log2-base to conduct the statistical analysis. The median age was 53, 46 (10.5%) patients had previous history of cardiac disease, MAC allo-HCTs were performed in 186 (42.6%) patients, and PTCY was administered in 242 (55.5%). The 1-year incidence of CAE was 12.6% (n = 55). The most prevalent cardiac events were heart failure and arrhythmias, 32.7% and 23.6% respectively, and the day +100 mortality rate of these patients was 40.5%. During the first 6 months after allo-HCT, EASIX trends were significantly higher in patients who developed CAE. Regression analyses confirmed that higher log2-EASIX values were predictors for higher risk for CAE during the first year after allo-HCT. This analysis identifies a significant association between higher endothelial activation, indirectly measured using EASIX, and higher risk for cardiac toxicity diagnosed during the first year after allo-HCT and extends the applicability of EASIX for identifying patients at risk for CAE.

Endothelial Activation and Stress Index in adults undergoing allogeneic hematopoietic cell transplantation with post-transplant cyclophosphamide-based prophylaxis.

BACKGROUND AIMS: Post-transplant cyclophosphamide (PTCY)-based prophylaxis is becoming widespread for allogeneic hematopoietic cell transplantation (allo-HCT) performed independently of the selected donor source. In parallel, use of the Endothelial Activation and Stress Index (EASIX)-considered a surrogate parameter of endothelial activation-for predicting patient outcomes and clinical complications is gaining popularity in the allo-HCT setting. METHODS: We first investigated whether the dynamics of EASIX after allo-HCT differ between patients receiving PTCY and patients receiving other prophylaxis. We then investigated whether the predictive capacity of EASIX persists in PTCY-based allo-HCT. A total of 328 patients transplanted between 2014 and 2020 were included, and 201 (61.2%) received PTCY. RESULTS: EASIX trends differed significantly between the groups. Compared with patients receiving other prophylaxis, patients receiving PTCY had lower EASIX on day 0 and higher values between day 7 and day 100. In patients receiving PTCY, higher EASIX correlated significantly with higher non-relapse mortality (NRM) and lower overall survival (OS) when measured before and during the first 180 days after allo-HCT. In addition, higher EASIX scores measured at specific time points were predictors of veno-occlusive disease (VOD), transplant-associated thrombotic microangiopathy (TA-TMA) and grade 2-4 acute graft-versus-host disease (aGVHD) risk. CONCLUSIONS: This study demonstrates how EASIX trends vary during the first 180 days after allo-HCT in patients receiving PTCY and those not receiving PTCY and validates the utility of this index for predicting NRM, OS and risk of VOD, TA-TMA and grade 2-4 aGVHD in patients receiving PTCY.

Easix Score Correlates With Endothelial Dysfunction Biomarkers and Predicts Risk of Acute Graft-Versus-Host Disease After Allogeneic Transplantation.

Plasma biomarkers of endothelial dysfunction have been postulated for the diagnosis and prognosis of acute graft-versus-host disease (aGVHD). However, their use is not validated in clinical practice yet. The endothelial activation and stress index (EASIX), a simple score based on routine laboratory parameters, is considered to be an indirect marker of endothelial damage. High value of EASIX was correlated with worse non-relapse mortality (NRM) and overall survival (OS) and a high risk of sinusoidal obstructive syndrome and transplant-associated thrombotic microangiopathy (TA-TMA). This study investigates the predictive value of plasma biomarkers and the EASIX score for the prediction of aGVHD. We assessed vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor receptor 1 (TNFR1), and VWF:Ag plasma levels and the EASIX score before allogeneic hematopoietic stem cell transplantation (allo-HSCT) and on days 0, 3, 7, 14, and 21 in an experimental cohort (n = 33). EASIX was transformed to a base-2 logarithm to perform the analysis. For the most relevant biomarkers, we estimate the optimal cutoff values and the discriminatory ability to differentiate patients with high-risk of aGVHD. The conclusions obtained in the experimental cohort were validated in a large cohort of 321 patients at the same institution. Plasma biomarkers and EASIX showed similar post-transplantation dynamics consisting of a progressive increase. Multivariate analysis showed an association between high TNFR1 levels and Log-2 EASIX score on day 7 after transplantation with an increased likelihood of developing aGVHD (hazard ratio [HR] = 1, P = .002; HR = 2.31, P = .013, respectively). Patients with TNFR1 ≥1300 ng/mL (HR = 7.19, P = .006) and Log2-EASIX ≥3 (HR = 14.7, P <.001) at day 7 after transplantation were more likely to develop aGVHD with high predictive accuracy (C-index of 74% and 81%, respectively). In the validation cohort, patients with Log2-EASIX ≥3 on day 7 after transplantation presented a significantly higher incidence of grade II-IV aGVHD (HR = 1.94, P = .004) independent of GVHD prophylaxis (HR = 0.38, P = .004), conditioning regimen (HR = 0.59, P =.02) and type of donor (HR = 2.38, P = .014). Differential degree of endothelial damage can be measured using both EASIX score and plasma biomarkers in the early post-transplantation period. Patients at risk of developing aGVHD could be easily identified by a high EASIX score. Both indicators of endothelial activation represent a promising approach to predict aGVHD.

Endothelial activation and damage as a common pathological substrate in different pathologies and cell therapy complications.

The endothelium is a biologically active interface with multiple functions, some of them common throughout the vascular tree, and others that depend on its anatomical location. Endothelial cells are continually exposed to cellular and humoral factors, and to all those elements (biological, chemical, or hemodynamic) that circulate in blood at a certain time. It can adapt to different stimuli but this capability may be lost if the stimuli are strong enough and/or persistent in time. If the endothelium loses its adaptability it may become dysfunctional, becoming a potential real danger to the host. Endothelial dysfunction is present in multiple clinical conditions, such as chronic kidney disease, obesity, major depression, pregnancy-related complications, septic syndromes, COVID-19, and thrombotic microangiopathies, among other pathologies, but also in association with cell therapies, such as hematopoietic stem cell transplantation and treatment with chimeric antigen receptor T cells. In these diverse conditions, evidence suggests that the presence and severity of endothelial dysfunction correlate with the severity of the associated disease. More importantly, endothelial dysfunction has a strong diagnostic and prognostic value for the development of critical complications that, although may differ according to the underlying disease, have a vascular background in common. Our multidisciplinary team of women has devoted many years to exploring the role of the endothelium in association with the mentioned diseases and conditions. Our research group has characterized some of the mechanisms and also proposed biomarkers of endothelial damage. A better knowledge would provide therapeutic strategies either to prevent or to treat endothelial dysfunction.

Functionalization of 3D printed polymeric bioresorbable stents with a dual cell-adhesive peptidic platform combining RGDS and YIGSR sequences.

Biomimetic surface modification with cell-adhesive peptides is a promising approach to improve endothelialization of current bioresorbable stents (BRS). Among them, RGDS and YIGSR sequences have been reported to mediate adhesion and migration of endothelial cells (ECs) while preventing platelet activation. This work presents the functionalization of novel 3D-printed poly-L-lactic acid (PLLA) and poly(L-lactic-co-ε-caprolactone) (PLCL) BRS with linear RGDS and YIGSR sequences, as well as a dual platform (PF) containing both motifs within a single biomolecule. Functionalized surfaces were characterized in terms of static contact angle, biomolecule distribution under confocal fluorescence microscopy and peptide quantification via detachment from the surface, showing a biomolecule density in the range of 0.5 to 3.5 nmol cm-2. Biological evaluation comprised a cell adhesion test on functionalized films with ECs and a blood perfusion assay on functionalized stents to assess ECs response and device hemocompatibility, respectively. Cell adhesion assays evidenced significantly increased cell number and spreading onto functionalized films with respect to control samples. Regarding stents' hemocompatibility, platelet adhesion onto PLCL stents was severely decreased with respect to PLLA. In addition, functionalization with RGDS, YIGSR and the PF rendered BRS stents displaying even further reduced platelet adhesion. In conclusion, the combination of intrinsically less prothrombogenic materials such as PLCL and its functionalization with EC-discriminating adhesive biomolecules paves the way for a new generation of BRS based on accelerated re-endothelialization approaches.

Characterization of the endotheliopathy, innate-immune activation and hemostatic imbalance underlying CAR-T cell toxicities: laboratory tools for an early and differential diagnosis.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell-based immunotherapy constitutes a revolutionary advance for treatment of relapsed/refractory hematological malignancies. Nevertheless, cytokine release and immune effector cell-associated neurotoxicity syndromes are life-threatening toxicities in which the endothelium could be a pathophysiological substrate. Furthermore, differential diagnosis from sepsis, highly incident in these patients, is challenging. Suitable laboratory tools could be determinant for their appropriate management. METHODS: Sixty-two patients treated with CAR-T cell immunotherapy for hematological malignancies (n=46 with CD19-positive diseases, n=16 with multiple myeloma) were included. Plasma samples were obtained: before CAR-T cell infusion (baseline); after 24-48 hours; at suspicion of any toxicity onset and 24-48 hours after immunomodulatory treatment. Biomarkers of endothelial dysfunction (soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble TNF receptor 1 (sTNFRI), thrombomodulin (TM), soluble suppression of tumorigenesis-2 factor (ST2), angiopoietin-2 (Ang-2)), innate immunity activation (neutrophil extracellular traps (NETs), soluble C5b-9 (sC5b-9)) and hemostasis/fibrinolysis (von Willebrand Factor antigen (VWF:Ag), ADAMTS-13 (A13), α2-antiplasmin (α2-AP), plasminogen activator inhibitor-1 antigen (PAI-1 Ag)) were measured and compared with those in cohorts of patients with sepsis and healthy donors. RESULTS: Patients who developed CAR-T cell toxicities presented increased levels of sVCAM-1, sTNFRI and ST2 at the clinical onset versus postinfusion values. Twenty-four hours after infusion, ST2 levels were good predictors of any CAR-T cell toxicity, and combination of ST2, Ang-2 and NETs differentiated patients requiring intensive care unit admission from those with milder clinical presentations. Association of Ang-2, NETs, sC5b-9, VWF:Ag and PAI-1 Ag showed excellent discrimination between severe CAR-T cell toxicities and sepsis. CONCLUSIONS: This study provides relevant contributions to the current knowledge of the CAR-T cell toxicities pathophysiology. Markers of endotheliopathy, innate immunity activation and hemostatic imbalance appear as potential laboratory tools for their prediction, severity and differential diagnosis.

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease, depending on steroid response.

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n = 4) and SS-aGVHD (n = 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p < 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.

Mafosfamide, a cyclophosphamide analog, causes a proinflammatory response and increased permeability on endothelial cells in vitro.

Post-transplantation cyclophosphamide (PTCy) has decreased GVHD incidence. Endothelial damage in allo-HCT is caused by multiple factors, including conditioning treatments and some immunosupressants, and underlies HCT-complications as GVHD. Nevertheless, the specific impact of PTCy on the endothelium remains unclear. We evaluated the effect of mafosfamide (MAF), an active Cy analog, on endothelial cells (ECs) vs. cyclosporine A (CSA), with known damaging endothelial effect. ECs were exposed to MAF and CSA to explore changes in endothelial damage markers: (i) surface VCAM-1, (ii) leukocyte adhesion on ECs, (iii) VE-cadherin expression, (iv) production of VWF, and (v) activation of intracellular signaling proteins (p38MAPK, Akt). Results obtained (expressed in folds vs. controls) indicate that both compounds increased VCAM-1 expression (3.1 ± 0.3 and 2.8 ± 0.6, respectively, p < 0.01), with higher leukocyte adhesion (5.5 ± 0.6, p < 0.05, and 2.8 ± 0.4, respectively). VE-cadherin decreased with MAF (0.8 ± 0.1, p < 0.01), whereas no effect was observed with CSA. Production of VWF augmented with CSA (1.4 ± 0.1, p < 0.01), but diminished with MAF (0.9 ± 0.1, p < 0.05). p38MAPK activation occurred with both compounds, being more intense and faster with CSA. Both drugs activated Akt, with superior MAF effect at longer exposure. Therefore, the cyclophosphamide analog MAF is not exempt from a proinflammatory effect on the endothelium, though without modifying the subendothelial characteristics.

Incidence, risk factors, and impact of early cardiac toxicity after allogeneic hematopoietic cell transplant.

This study investigates early cardiac events (ECEs) occurring during the first 180 days after allogeneic hematopoietic cell transplant (allo-HCT) in 416 adults receiving posttransplant cyclophosphamide (PTCY) (n = 258) or not receiving PTCY (n = 158). Total body irradiation (TBI) was given to 133 (31.9%) patients, of whom 111 (83.4%) received TBI combined with PTCY. The day +180 cumulative incidence function (CIF) of ECEs was 8.4%, with heart failure (n = 13) and pericardial complications (n = 11) being the most prevalent complications. The incidence of ECEs was higher in patients receiving PTCY, and receiving TBI. ECEs were more prevalent in haploidentical HCTs than in matched sibling donor, 10/10 HLA-matched unrelated donor, and 9/10 HLA-mismatched unrelated donor allo-HCTs. As for the ECE risk from the combination of PTCY and TBI, the multivariate analysis reported that patients receiving PTCY without TBI, TBI without PTCY, and TBI with PTCY were at higher risk for ECEs compared with patients receiving neither PTCY nor TBI. Pre-existing cardiac morbidity predicted ECEs. However, using high-dose CY-containing preparative regimens did not increase the risk for cardiac toxicity at +180 days after allo-HCT. ECEs were associated with higher nonrelapse mortality and lower overall survival. Considering that PTCY and TBI were predictors for ECEs, and the impact of this complication on transplant mortality, the implementation of cardiac monitoring plans could be appropriate in patients receiving these medications.

Early vascular endothelial complications after hematopoietic cell transplantation: Role of the endotheliopathy in biomarkers and target therapies development.

This work aims to review the role of endothelial dysfunction underlying the main complications appearing early after autologous and allogeneic hematopoietic cell transplantation (HCT). The endothelial damage as the pathophysiological substrate of sinusoidal obstruction syndrome (SOS) is well established. However, there is growing evidence of the involvement of endothelial dysfunction in other complications, such as acute graft-versus-host disease (aGVHD) and transplant-associated thrombotic microangiopathy (TA-TMAs). Moreover, HCT-related endotheliopathy is not only limited to the HCT setting, as there is increasing evidence of its implication in complications derived from other cellular therapies. We also review the incidence and the risk factors of the main HCT complications and the biological evidence of the endothelial involvement and other linked pathways in their development. In addition, we cover the state of the art regarding the potential use of the biomarkers of endotheliopathy in the prediction, the early diagnosis, and the follow-up of the HCT complications and summarize current knowledge points to the endothelium and the other linked pathways described as potential targets for the prevention and treatment of HCT-complications. Lastly, the endothelium-focused therapeutic strategies that are emerging and might have a potential impact on the survival and quality of life of post-HCT-patients are additionally reviewed.

Differences and similarities in endothelial and angiogenic profiles of preeclampsia and COVID-19 in pregnancy.

BACKGROUND: COVID-19 presents a spectrum of signs and symptoms in pregnant women that might resemble preeclampsia. Differentiation between severe COVID-19 and preeclampsia is difficult in some cases. OBJECTIVE: To study biomarkers of endothelial damage, coagulation, innate immune response, and angiogenesis in preeclampsia and COVID-19 in pregnancy in addition to in vitro alterations in endothelial cells exposed to sera from pregnant women with preeclampsia and COVID-19. STUDY DESIGN: Plasma and sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) and from women with normotensive pregnancies as controls (n=10) and patients with preeclampsia (n=13). A panel of plasmatic biomarkers was assessed, including vascular cell adhesion molecule-1, soluble tumor necrosis factor-receptor I, heparan sulfate, von Willebrand factor antigen (activity and multimeric pattern), α2-antiplasmin, C5b9, neutrophil extracellular traps, placental growth factor, soluble fms-like tyrosine kinase-1, and angiopoietin 2. In addition, microvascular endothelial cells were exposed to patients' sera, and changes in the cell expression of intercellular adhesion molecule 1 on cell membranes and von Willebrand factor release to the extracellular matrix were evaluated through immunofluorescence. Changes in inflammation cell signaling pathways were also assessed by of p38 mitogen-activated protein kinase phosphorylation. Statistical analysis included univariate and multivariate methods. RESULTS: Biomarker profiles of patients with mild COVID-19 were similar to those of controls. Both preeclampsia and severe COVID-19 showed significant alterations in most circulating biomarkers with distinctive profiles. Whereas severe COVID-19 exhibited higher concentrations of vascular cell adhesion molecule-1, soluble tumor necrosis factor-α receptor I, heparan sulfate, von Willebrand factor antigen, and neutrophil extracellular traps, with a significant reduction of placental growth factor compared with controls, preeclampsia presented a marked increase in vascular cell adhesion molecule-1 and soluble tumor necrosis factor-α receptor I (significantly increased compared with controls and patients with severe COVID-19), with a striking reduction in von Willebrand factor antigen, von Willebrand factor activity, and α2-antiplasmin. As expected, reduced placental growth factor, increased soluble fms-like tyrosine kinase-1 and angiopoietin 2, and a very high soluble fms-like tyrosine kinase-1 to placental growth factor ratio were also observed in preeclampsia. In addition, a significant increase in C5b9 and neutrophil extracellular traps was also detected in preeclampsia compared with controls. Principal component analysis demonstrated a clear separation between patients with preeclampsia and the other groups (first and second components explained 42.2% and 13.5% of the variance), mainly differentiated by variables related to von Willebrand factor, soluble tumor necrosis factor-receptor I, heparan sulfate, and soluble fms-like tyrosine kinase-1. Von Willebrand factor multimeric analysis revealed the absence of von Willebrand factor high-molecular-weight multimers in preeclampsia (similar profile to von Willebrand disease type 2A), whereas in healthy pregnancies and COVID-19 patients, von Willebrand factor multimeric pattern was normal. Sera from both preeclampsia and severe COVID-19 patients induced an overexpression of intercellular adhesion molecule 1 and von Willebrand factor in endothelial cells in culture compared with controls. However, the effect of preeclampsia was less pronounced than the that of severe COVID-19. Immunoblots of lysates from endothelial cells exposed to mild and severe COVID-19 and preeclampsia sera showed an increase in p38 mitogen-activated protein kinase phosphorylation. Patients with severe COVID-19 and preeclampsia were statistically different from controls, suggesting that both severe COVID-19 and preeclampsia sera can activate inflammatory signaling pathways. CONCLUSION: Although similar in in vitro endothelial dysfunction, preeclampsia and severe COVID-19 exhibit distinctive profiles of circulating biomarkers related to endothelial damage, coagulopathy, and angiogenic imbalance that could aid in the differential diagnosis of these entities.

An endothelial proinflammatory phenotype precedes the development of the engraftment syndrome after autologous Hct.

Engraftment syndrome (ES) is a common complication after autologous hematopoietic cell transplantation (auto-HCT) whose pathophysiological substrate remains unclear. We investigated whether endothelial damage could contribute to ES. Circulating ECs-damage biomarkers were measured in plasma from patients with (ES; n = 14) or without ES (non-ES; n = 20), collected at different time points: before HCT, 5 (S5) and 10 days (S10) after HCT, and at either the ES onset (SON) or the discharge day (SDIS). Also, cultured endothelial cells (ECs) were exposed to serum samples, obtained at the same points, to evaluate changes in ECs-activation (ICAM-1, VE-Cadherin) biomarkers, the reactivity of ECs towards leukocytes, and activation of intracellular signaling proteins related to inflammation (p38MAPK) and proliferation (Erk1/2). Results showed that circulating VWF, sTNFR1 and sVCAM-1 levels were higher in ES patients at all the points assessed, especially at SON. In vitro results showed an increased ICAM-1 expression on ECs exposed to ES samples vs. non-ES samples, especially to S5, with elevated leukocyte adhesion. Also, a lower VE-Cadherin expression and an increased phosphorylation of p38MAPK and Erk1/2 proteins were observed in ECs exposed to ES vs. non-ES samples. Our results indicate that endothelial activation precedes ES development and could be one of its pathophysiological substrates.

Distinctive Biomarker Features in the Endotheliopathy of COVID-19 and Septic Syndromes.

BACKGROUND: Endotheliopathy is a key element in COVID-19 pathophysiology, contributing to both morbidity and mortality. Biomarkers distinguishing different COVID-19 phenotypes from sepsis syndrome remain poorly understood. OBJECTIVE: To characterize circulating biomarkers of endothelial damage in different COVID-19 clinical disease stages compared with sepsis syndrome and normal volunteers. METHODS: Patients with COVID-19 pneumonia (n = 49) were classified into moderate, severe, or critical (life-threatening) disease. Plasma samples were collected within 48 to 72 h of hospitalization to analyze endothelial activation markers, including soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), von Willebrand Factor (VWF), A disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 (ADAMTS-13) activity, thrombomodulin (TM), and soluble TNF receptor I (sTNFRI); heparan sulfate (HS) for endothelial glycocalyx degradation; C5b9 deposits on endothelial cells in culture and soluble C5b9 for complement activation; circulating dsDNA for neutrophil extracellular traps (NETs) presence, and α2-antiplasmin and PAI-1 as parameters of fibrinolysis. We compared the level of each biomarker in all three COVID-19 groups and healthy donors as controls (n = 45). Results in critically ill COVID-19 patients were compared with other intensive care unit (ICU) patients with septic shock (SS, n = 14), sepsis (S, n = 7), and noninfectious systemic inflammatory response syndrome (NI-SIRS, n = 7). RESULTS: All analyzed biomarkers were increased in COVID-19 patients versus controls (P < 0.001), except for ADAMTS-13 activity that was normal in both groups. The increased expression of sVCAM-1, VWF, sTNFRI, and HS was related to COVID-19 disease severity (P < 0.05). Several differences in these parameters were found between ICU groups: SS patients showed significantly higher levels of VWF, TM, sTNFRI, and NETS compared with critical COVID-19 patients and ADAMTS-13 activity was significantly lover in SS, S, and NI-SIRS versus critical COVID-19 (P < 0.001). Furthermore, α2-antiplasmin activity was higher in critical COVID-19 versus NI-SIRS (P < 0.01) and SS (P < 0.001), whereas PAI-1 levels were significantly lower in COVID-19 patients compared with NI-SIRS, S, and SS patients (P < 0.01). CONCLUSIONS: COVID-19 patients present with increased circulating endothelial stress products, complement activation, and fibrinolytic dysregulation, associated with disease severity. COVID-19 endotheliopathy differs from SS, in which endothelial damage is also a critical feature of pathobiology. These biomarkers could help to stratify the severity of COVID-19 disease and may also provide information to guide specific therapeutic strategies to mitigate endotheliopathy progression.

Complement as the enabler of carfilzomib-induced thrombotic microangiopathy.

Carfilzomib has been associated with the development of thrombotic microangiopathy (TMA) in relapsed/refractory multiple myeloma patients, a severe disease with no currently available aetiological treatment. We evaluated the potential role of terminal complement pathway in four patients with carfilzomib-induced TMA. Membrane attack complex (C5b-9) deposition on endothelial cells in culture exposed to plasma from patients during the acute phase of the disease suggests complement overactivation as a mechanism of potential endothelial damage in three out of four patients. If confirmed in larger cohorts, C5b-9 evaluation will allow early identification of patients who could benefit from complement blockade and treatment monitoring.

The avoidance of G-CSF and the addition of prophylactic corticosteroids after autologous stem cell transplantation for multiple myeloma patients appeal for the at-home setting to reduce readmission for neutropenic fever.

BACKGROUND: Autologous stem cell transplantation (ASCT) remains the standard of care for young multiple myeloma (MM) patients; indeed, at-home ASCT has been positioned as an appropriate therapeutic strategy. However, despite the use of prophylactic antibiotics, neutropenic fever (NF) and hospital readmissions continue to pose as the most important limitations in the outpatient setting. It is possible that the febrile episodes may have a non-infectious etiology, and engraftment syndrome could play a more significant role. The aim of this study was to analyze the impact of both G-CSF withdrawal and the addition of primary prophylaxis with corticosteroids after ASCT. METHODS: Between January 2002 and August 2018, 111 MM patients conditioned with melphalan were managed at-home beginning +1 day after ASCT. Three groups were established: Group A (n = 33) received standard G-CSF post-ASCT; group B (n = 32) avoided G-CSF post-ASCT; group C (n = 46) avoided G-CSF yet added corticosteroid prophylaxis post-ASCT. RESULTS: The incidence of NF among the groups was reduced (64%, 44%, and 24%; P<0.001), with a non-significant decrease in hospital readmissions as well (12%, 6%, and 2%; P = 0.07). The most important variables identified for NF were: HCT-CI >2 (OR 6.1; P = 0.002) and G-CSF avoidance plus corticosteroids (OR 0.1; P<0.001); and for hospital readmission: age ≥60 years (OR 14.6; P = 0.04) and G-CSF avoidance plus corticosteroids (OR 0.07; P = 0.05). CONCLUSIONS: G-CSF avoidance and corticosteroid prophylaxis post ASCT minimize the incidence of NF in MM patients undergoing at-home ASCT. This approach should be explored in a prospective randomized clinical trial.

Defibrotide inhibits donor leucocyte-endothelial interactions and protects against acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapy for the treatment of high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. Acute graft-versus-host disease (aGvHD) remains the most frequent cause of non-relapse mortality following allo-HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi-target drug successfully employed for prophylaxis and treatment of veno-occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo-HCT. Using a fully MHC-mismatched murine model of allo-HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD-associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo-HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte-endothelial interactions by down-regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo-HCT, paving the way for new therapeutic approaches.

The induction strategies administered in the treatment of multiple myeloma exhibit a deleterious effect on the endothelium.

Multiple myeloma induction treatment includes proteasome inhibitors (PI) and immunomodulatory agents at present. The incidence of engraftment syndrome, a transplant complication potentially related to endothelium, has increased in the last years. Our aim was to investigate whether bortezomib (Velcade, V), thalidomide (T), and dexamethasone (D) affect the endothelium, and explore defibrotide (DF) as protective agent. Endothelial cells (ECs) in culture were exposed to the compounds separately or in combination, without (VTD) and with DF (VTD + DF). Changes in markers of: (i) inflammation (ICAM-1 expression and leukocyte adhesion), (ii) VWF production, (iii) cell permeability (VE-cadherin expression and cell monolayer integrity), and (iv) oxidative stress (ROS production and eNOS expression) were measured. ICAM-1 and VWF expression increased significantly in VTD but were similar to controls in VTD + DF. Separately, bortezomib was the main deleterious agent whereas dexamethasone showed no harmful effect. Leukocyte adhesion showed similar trends. VE-cadherin expression was lower in VTD and normalized in VTD + DF. EC permeability increased only with bortezomib. No changes were observed in oxidative stress markers. Our results demonstrate that bortezomib damages the endothelium, and DF prevents this effect. A better knowledge of the induction drugs impact will allow the design of measures to protect the endothelium.