Human Bone Marrow Endothelial Cells Research Articles

Conventional Ultrasound, Immunohistochemical Factors and BRAFV600E Mutation in Predicting Central Cervical Lymph Node Metastasis of Papillary Thyroid Carcinoma

The study was aimed at evaluating the correlation between central cervical lymph node metastasis (CLNM) in papillary thyroid carcinoma (PTC) patients and ultrasound (US) features, immunohistochemical factors and BRAFV600E mutation. A total of 225 consecutive patients (225 PTCs) who had undergone surgery were included. All PTCs were pre-operatively analysed by US with respect to size, components, echogenicity, shape, margins, microcalcification, multiple cancers or not, internal vascularity and capsule contact or involvement. The presence of four immunohistochemical factors, including cytokeratin 19, human bone marrow endothelial cell 1, galectin-3 and thyroid peroxidase, and BRAFV600E mutation was also evaluated. Univariate and multivariate analyses were performed to identify the risk factors for central CLNM, and a risk model was established. Pathologically, 44% (99/225) of the PTCs had central CLNMs. Multivariate analysis revealed that size ≤10mm, microcalcification, internal vascularity, capsule contact or involvement and BRAFV600E mutation were independent risk factors for central CLNM. The risk score for central CLNM was calculated as follows: risk score = 1.5 × (if lesion size ≤10 mm) + 1.9 × (if microcalcification) + 0.8 × (if internal flow) + 3.0 × (if capsule contact or involvement) + 1.5 × (if BRAFV600E mutation). The rating result was divided into six stages, and the relevant risk rates of central CLNM were 0% (0/1), 0% (0/22), 7.4% (4/54), 48.6% (34/70), 71.2% (42/59) and 100% (19/19), respectively. In conclusion, PTC ≤10mm, microcalcification, internal vascularity, capsule contact or involvement and BRAFV600E mutation are risk factors for central CLNM. The risk model may be useful in treatment planning and management of patients with PTCs.

Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors.

The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.

Potential Role of Humoral IL-6 Cytokine in Mediating Pro-Inflammatory Endothelial Cell Response in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial disease with limited therapeutic options. Numerous intrinsic and extrinsic factors are involved in ALS motor neuron degeneration. One possible effector accelerating motor neuron death in ALS is damage to the blood-Central Nervous System barrier (B-CNS-B), mainly due to endothelial cell (EC) degeneration. Although mechanisms of EC damage in ALS are still unknown, vascular impairment may be initiated by various humoral inflammatory factors and other mediators. Systemic IL-6-mediated inflammation is a possible early extrinsic effector leading to the EC death causing central nervous system (CNS) barrier damage. In this review, we discuss the potential role of humoral factors in triggering EC alterations in ALS. A specific focus was on humoral IL-6 cytokine mediating EC inflammation via the trans-signaling pathway. Our preliminary in vitro studies demonstrated a proof of principle that short term exposure of human bone marrow endothelial cells to plasma from ALS patient leads to cell morphological changes, significantly upregulated IL-6R immunoexpression, and pro-inflammatory cell response. Our in-depth understanding of specific molecular mechanisms of this humoral cytokine in EC degeneration may facilitate an endothelial-IL-6-targeting therapy for restoring cell homeostasis and eventually reestablishing B-CNS-B integrity in ALS.

HBME-1 expression in differentiated thyroid carcinoma and its correlation with the ultrasonic manifestation of thyroid.

We investigated expression of the protein human bone marrow endothelial cell marker-1 (HBME-1) in differentiated thyroid carcinoma tissues, and analyzed its correlation with ultrasonic manifestation of thyroid. The immunohistochemistry (IHC) staining method was used to measure the expression of HBME-1 in patient with nodular goiter (control group), papillary differentiated thyroid carcinoma (papillary carcinoma group) and follicular differentiated thyroid carcinoma (follicular carcinoma group) to investigate the differences in expression of HBME-1. We further analyzed the correlation of the expression of HBME-1 in the papillary carcinoma group and the follicular carcinoma group with ultrasonic manifestation of thyroid. In both the papillary carcinoma group and the follicular carcinoma group, the levels of HBME-1 in affected tissues and the IHS score of HBME-1 expression were higher than those in the control group (p<0.05). In the papillary carcinoma group, the mean IHS score of HBME-1 expression in affected tissues was higher than in the follicular carcinoma group (p<0.05). There were no statistically significant differences in comparison to HBME-1 expression in affected tissues between the papillary carcinoma group and the follicular carcinoma group (p>0.05). Between the papillary carcinoma group and the follicular carcinoma group, the differences in the comparison of the nodule diameter, echo, shape, boundary, calcification and blood flow signal were statistically significant (p<0.05), but incidence rate of enlargement of cervical lymph nodules between the groups were not statistically significant (p>0.05). Among patients in the papillary carcinoma group, the comparison of the nodule diameter, echo, shape, boundary, calcification and blood flow signal between the HBME-1-positive patients and the HBME-1-negative patients showed no statistical significance (p>0.05), but in the nodules of HBME-1-positive patients, the proportion of blood flow signal was higher than that in the nodules of HBME-1-negative patients. Among patients in the follicular carcinoma group, there was no statistically significant differences in the comparison of ultrasonic manifestation of thyroid (p>0.05). Therefore, there are difference in HBME-1 expression and ultrasonic manifestations of thyroid in patients with papillary carcinoma and follicular differentiated thyroid carcinoma.

Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering.

The conventional tissue engineering is based on seeding of macroporous scaffold on its surface ("top-down" approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on "bottom-up" approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

Utilization of mechano‐biological models to predict cell adhesion interactions between bone marrow endothelial cells and breast cancer cells.

The purpose of our study was to produce a mechano‐biological model that could characterize and predict adhesive interactions between human breast cancer cells (MB435) and human bone marrow endothelial cells (HBME). Bone is a common site of metastasis for breast cancer and the mechanisms of metastasis are not fully characterized. In this study we have combined experimental and modelling approaches to probe interactions of human bone marrow endothelial cells (HBMEC‐60) with breast cancer cells (MB435). Specifically, atomic force microscopy (AFM) was used to measure adhesion forces between a single MB435 cell (attached to the AFM tip) and a monolayer of HBMEC‐60 cells for differing times of contact. In addition, we developed a stochastic model of the individual adhesive interactions that occur between the two cell‐types to mimic the AFM experimental protocol. This was accomplished by formulating a 2D discrete stochastic‐elastic computational model to study the adhesion binding dynamics that underpin cell‐cell attachment. The model allows detailed quantification in binding dynamics and the location, lifetime, and strength of cell‐cell adhesions. To describe cell‐cell interactions, we considered the following components within a discrete stochastic computational framework: intracellular actin in each cell and adhesion proteins in the cell membranes in 4 possible states: freely diffusing, actin‐bound to the first cell, actin‐bound to the second cell and fully‐bound to cytoskeleton within both cells. Adhesions between the cytoskeleton networks of the two cells were connected to form a network of springs according to the transitions, which obey stochastic binding/unbinding events. Free adhesion molecules diffused via a random walk. The dynamically changing network structure was coupled to the local mechanical environment through force‐dependent binding/unbinding propensities. Experimentally, we found that peak adhesion forces increased with increasing contact time between the MB435 and HBMEC‐60 cells and that total adhesion force increased dramatically within the first 30 sec of contact which then gradually plateaued. These experimental data demonstrate a strong dependence of MB435‐HBME adhesion strength on cell‐cell contact time. Additionally, we found that our stochastic computational model recapitulated these experimental findings, suggesting that important mechanisms have been captured in the model. This validates our experiment vs. model approach and is therefore allowing us to interrogate the model further to derive further insights into the cell‐cell interactions observed here.

Intravenously Transplanted Human Bone Marrow Endothelial Progenitor Cells Engraft Within Brain Capillaries, Preserve Mitochondrial Morphology, and Display Pinocytotic Activity Toward Blood-Brain Barrier Repair in Ischemic Stroke Rats.

Stroke is a life-threatening disease with limited therapeutic options. Cell therapy has emerged as an experimental stroke treatment. Blood-brain barrier (BBB) impairment is a key pathological manifestation of ischemic stroke, and barrier repair is an innovative target for neurorestoration in stroke. Here, we evaluated via electron microscopy the ability of transplanted human bone marrow endothelial progenitor cells (hBMEPCs) to repair the BBB in adult Sprague-Dawley rats subjected to transient middle cerebral artery occlusion (tMCAO). β-galactosidase prelabeled hBMEPCs were intravenously transplanted 48 hours post-tMCAO. Ultrastructural analysis of microvessels in nontransplant stroke rats revealed typical BBB pathology. At 5 days post-transplantation with hBMEPCs, stroke rats displayed widespread vascular repair in bilateral striatum and motor cortex, characterized by robust cell engraftment within capillaries. hBMEPC transplanted stroke rats exhibited near normal morphology of endothelial cells (ECs), pericytes, and astrocytes, without detectable perivascular edema. Near normal morphology of mitochondria was also detected in ECs and perivascular astrocytes from transplanted stroke rats. Equally notable, we observed numerous pinocytic vesicles within engrafted cells. Robust engraftment and intricate functionality of transplanted hBMEPCs likely abrogated stroke-altered vasculature. Preserving mitochondria and augmenting pinocytosis in cell-based therapeutics represent a new neurorestorative mechanism in BBB repair for stroke. Stem Cells 2017;35:1246-1258.

Variation of Red Blood Cell Rheology and Adhesiveness during Vaso-Occlusive Crises in Sickle Cell Patients

Introduction: Impaired red blood cell (RBC) rheology, increased RBC adhesiveness to the vascular wall, enhanced inflammation and blunted vascular reactivity are involved in the pathophysiology of sickle cell anemia (SCA). Painful vaso-occlusive crisis (VOC) is the most frequent complication encountered by SCA patients. While several studies compared several biomarkers of severity between patients at steady state and others during VOC, very few works compared the same patients in the two conditions. It is therefore difficult to know what happens during VOC. The present study was devoted to compare several hematological, biochemical, and rheological parameters, as well as RBC adhesiveness at steady state and during VOC. Altogether, 36 SCA patients were studied.Methods: This prospective monocentric study was performed at the University Hospital of Pointe-a-Pitre (Guadeloupe, French West Indies), in accordance with the guidelines set by the declaration of Helsinki and was approved by the Regional Ethics Committee (CPP Sud/Ouest Outre Mer III, Bordeaux, France, registration number: 2012-A00701-42). After admission to the emergency department for a VOC episode, patients were informed about the purpose and procedures of the study and gave their written consent. Blood was sampled at the arrival of the patients at the emergency department before they received any medications. A visit to the Sickle Cell Center was then scheduled at least 3 months after the emergency department admission to collect routine blood samples at steady state. Steady state condition was defined as a period free of blood transfusion in the previous three months and without any acute SCA complications in the previous two months. RBC deformability was determined at 3 and 30 Pa by ektacytometry (LORRCA, Mechatronics), RBC aggregation properties (RBC aggregation and RBC disaggregation threshold) by syllectometry (LORRCA), blood viscosity by cone-plate viscosimetry (Brookfield, DVII+ model with CPE 40 spindle) at 225 s-1. Irreversible sickle cells (ISCs) were measured on an Imagestream ISX MkII flow cytometer (Amnis Corp, EMD Millipore). Lu/BCAM, ICAM-4/Lw and the alpha4-beta1 integrin were measured by flow cytometry at the RBC surface (FACSCanto II, BD Biosciences). RBC adhesion to monolayers of transformed human bone marrow endothelial cells (TrHBMECs) was studied in continuous flow conditions in Vena8 Endothelial+ Biochips (Cellix Ltd). Other hematological and biochemical parameters were measured by standard techniques.Results: Compared to steady state values, white blood cell (9.2 [6.7-10.6] versus 12.3 [10.1-16.2] 109/L, p < 0.001) and C-Reactive Protein (3.7 [3.3-6.0] versus 7.1 [3.3-17.5] mg/L, p < 0.05) levels increased during VOC (table 1). Lactate dehydrogenase level slightly increased during VOC (418 [351-564] versus 437 [370-727] IU/L, p < 0.03) but no change was observed for hemoglobin. RBC deformability slightly decreased during VOC (0.34 [0.26-0.44]) compared to steady state (0.38 [0.31-0.46], p < 0.02). RBC aggregation increased during VOC (55 [46-60 %] compared to steady state (51 [46-54] %, p < 0.05). No difference was detected for blood viscosity, RBC surface proteins, RBC adhesion, and RBC disaggregation threshold between the two conditions. During VOC, the percentage of ISCs was inversely correlated with deformability (p < 0.002 and p < 0.006 at 3 and 30 Pa, respectively), but positively correlated with RBC disaggregation threshold (p < 0.002) and with RBC adhesion to TrHBMECs (p < 0.008) (figure 1). At steady state, it was significantly correlated only with the disaggregation threshold (positive correlation, p < 0.03).Discussion: The most striking observation of this study is the positive correlation between ISCs (rigid cells) and RBC adhesion properties during overt VOC. This is in contrast with the general observation that the most deformable RBCs are those exhibiting the strongest adhesiveness and possibly involved in VOC initiation. Strengthened RBC aggregates may also disturb the blood flow into the microcirculation, hence participating to VOC progression and sustention. Drugs targeting RBC deformability (ISCs) and RBC aggregates might be helpful during established VOC in SCA. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Screening of differentially expressed protein kinases in bone marrow endothelial cells and the protective effects of the p38a inhibitor SB203580 on bone marrow in liver fibrosis.

Hematological abnormalities are frequently observed in patients with liver cirrhosis (LC). A previous study demonstrated that the apoptosis and damage of endothelial cells could cause the hematological abnormalities in LC. Protein kinases are one of the most important factors that regulate cell behavior, and are potential therapeutic targets for the treatment of a number of diseases. In a previous study, whole genome profiling was used to identify differentially expressed genes in human bone marrow endothelial cells treated with serum from 26 patients with LC. From this data set, the present study identified 14 differentially expressed kinase genes in human bone marrow endothelial cells in LC from the microarray data, including p38a, AKT1 and PDK1. Pathway analysis revealed that these kinase genes were enriched in certain important LC-associated pathways (e.g. MAPK and WNT signaling pathway). Literature mining revealed that p38a was associated with bone marrow apoptosis; therefore, p38a and its inhibitor, SB203580, were selected as potential therapeutic targets in the present study. The results of hematoxylin-eosin and Masson's trichrome staining of livers from a rat model of liver fibrosis (LF) that underwent ligation of the bile duct demonstrated that SB203580 reduced the degree of LF. In addition, SB203580-treated rats with LF demonstrated a significantly higher number of platelets when compared with the untreated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis indicated that apoptosis of bone marrow tissue in rats with LF was inhibited by SB203580. In addition, the results from the immunohistochemical analysis demonstrated that SB203580 reduced the expression of von Willebrand factor and caspase 3 in the bone marrow of rats with LF. In conclusion, the results from the present study indicate that the p38a kinase inhibitor, SB203580, may exhibit a protective effect on bone marrow tissues in rats with LF. This suggests that protein kinases and their inhibitors may present novel therapeutic strategies for the treatment of hematological abnormalities in patients with LC.

Clinical significance of HBME-1, Galectin-3, and CK19 expression and the status of BRAF mutation in papillary thyroid carcinoma

Abstract Objective The aim of this study was to explore the clinical significance of the expression of proteins human bone marrow endothelial cell markers (HBME-1), Galectin-3, and cytokeratin19 (CK19), as well as the status of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation in papillary thyroid carcinoma (PTC). Methods Immunohistochemical staining was performed in 82 specimens each of PTC and papillary benign lesions to detect the expression of HBME-1, Galectin-3, and CK19. Polymerase chain reaction (PCR) and gene sequencing were performed on 60 specimens each of PTC and papillary benign lesions to detect the status of BRAF mutation. Results The positive expression ratios of HBME-1, Galectin-3, and CK19 in PTC were 98.8%, 97.6% and 100% respectively, which were significantly higher than the expressions in papillary benign lesions (P &lt; 0.05). No significant relationship was observed between the expression of these makers and the clinicopathological features of PTC. The sensitivity of co-expression of HBME-1 and CK19 or HBME-1 and Galectin-3 as diagnostic criteria of PTC was 99.9%, with a specificity of 95.4%. BRAF mutation was detected in 40 of 60 PTC (66.7%) specimens. There was a statistical difference in BRAF mutations between PTC and papillary benign lesions (P &lt; 0.05); there were no associations between BRAF mutation and the clinicopathological features of PTC. Conclusion Combined immunohistochemical staining of HBME-1, Galectin-3, and CK19 can further improve the sensitivity and specificity of differential diagnosis of PTC. BRAF mutation is a significant genetic event, which may have diagnostic value for PTC.

Endothelial cell derived angiocrine support of acute myeloid leukemia targeted by receptor tyrosine kinase inhibition

In acute myeloid leukemia (AML), refractory disease is a major challenge and the leukemia microenvironment may harbor refractory disease. Human AML cell lines KG-1 and HL-60 expressed receptors also found on endothelial cells (ECs) such as VEGFRs, PDGFRs, and cKit. When human AML cells were co-cultured with human umbilical vein endothelial cells (HUVECs) and primary bone marrow endothelial cell (BMECs), the AML cells were more resistant to cytarabine chemotherapy, even in transwell co-culture suggesting angiocrine regulation. Primary BMECs secreted significantly increased levels of VEGF-A and PDGF-AB after exposure to cytarabine. Pazopanib, a receptor tyrosine kinase inhibitor (RTKI) of VEGFRs, PDGFRs, and cKit, removed EC protection of AML cells and enhanced AML cell sensitivity to cytarabine. Xenograft modeling showed significant regression of AML cells and abrogation of BM hypervascularity in RTKI treated cohorts. Together, these results show direct cytotoxicity of RTKIs on AML cells and reversal of EC protection. Combining RTKIs with chemotherapy may serve as promising therapeutic strategy for patients with AML.

A Furan-Based Lewis-Y-(CD174)-Saccharide Mimetic Inhibits Endothelial Functions and In Vitro Angiogenesis.

Angiogenesis is afundamental process underlying cancer progression and autoimmune disease. Lewis Yis known as aregulated glycan-structure supporting human endothelial function and angiogenesis. We hypothesize that Lewis Ybased analogues interfere with Lewis Ymediated endothelial functions and angiogenesis. We therefore evaluated the ability of 3, 4-bis [(b-D-galactopyranosyl)osy]-methyl-furan (BGF) afuran-based Lewis-Y saccharide mimetic to inhibit human endothelial adhesion, migration and in vitro angiogenesis. The ability of BGF and additional furan-based saccharide-mimetics was investigated to inhibit adhesion and migration of human bone marrow endothelial cells (HBMEC). Influence of BGF was tested on amulticelluar in vitro -angiogenesis assay in the presence of VEGF. BGF significantly inhibited HBMEC adhesion and migration stimulated by TNF-alpha by up to 70%. The anti-adhesive effect of BGF was particularly evident when HBMEC adhesion and migration was tested on collagen as extracellular matrix with weaker effect when laminin and fibronectin were used as an extracellular matrix. BGF was ineffective when HBMEC were stimulated with VEGF. The inhibition of endothelial function translated into asignificant inhibitory effect of BGF in the multicellular in vitro angiogenesis-assay. BGF reduced the angiogenesis index compared to the positive controls by 32%. We identified the ability of the furan-based Lewis Ysaccharide mimetic BGF as aspecific modulator of TNF-alpha activated endothelial function and in vitro angiogenesis. BGF and other related glycan analogues should further be explored for their ability to down modulate endothelial activation in TNF-alpha driven pathophysiologic conditions in autoimmune disease and cancer indications.

Chemosensitizing Leukemia By Targeting the Leukemia Microenvironment with Vascular Disrupting Combretastatins

Background: Refractory disease is the greatest challenge in treating leukemia. We recently discovered that blood vessels serve as a protective sanctuary for acute myeloid leukemia (AML) and developed a novel combretastatin, OXi4503, to disrupt this vascular niche. We translated our findings into a phase I clinical study for patients with refractory AML (ClinicalTrials.gov NCT01085656). An interim analysis of this trial suggested that optimal treatment of AML will likely require more than a single microenvironment targeting agent. Other AML trials, such as those with single agent sunitinib, bevacizumab, or pazopanib also support this concept. Therefore, we examined the effects of combining microenvironment targeting agents with cytotoxic chemotherapy.Methods: Bone marrow endothelial cells (BMECs) were isolated from bone marrow of healthy individuals. In vitro co-cultures of human AML (KG1) and BMECs were used to examine various treatments with cytarabine (Ara-C), azacitidine, and vascular disrupting combretastatins (CA1 and CA4). Cell death was analyzed by PI staining and flow cytometry. Cell cycle distribution was analyzed using 7-AADand BrdU staining. Given that combretastatins bind reversibly to B-tubulin, immunostaining of treated cells was performed using antibodies to B-tubulin and VCAM-1 and analyzed by fluorescence microscopy. Finally, the drug combinations with optimal in vitro effects were then tested in vivo using a xenograft model of human AML.Results: Co-culturing AML cells with BMECs afforded the leukemia cells a protective survival advantage despite treatments with Ara-C or azacitidine, further supporting the evolving understanding that leukemia cells located in or near the vascular niche may be a source of refractory disease. BMECs treated with combretastatins showed distinct degradation and disruption of microtubule cytoskeleton as well as an internalization of the adhesion molecule VCAM-1 one hour after combretastatin treatment. The combretastatin-treated BMECs were still viable but showed dramatic reduction in migratory function and adhesion molecule expression. When AML cells were co-cultured on combretastatin-treated BMECs, leukemia cell adhesion was significantly reduced. Treatment with chemotherapy agents, Ara-C or azacitidine, in co-cultures of AML + combretastatin-treated BMECs showed significant reductions in AML cell viability (Ara-C: 71.4% (Vehicle) vs 54.4% (CA1) and 59% (CA4), p < .001; azacitidine: 73.5% (Vehicle) vs 56.7% (CA1) and 59.2% (CA4), p < .001), suggesting that combretastatin pre-treatment reverses EC protection of AML cells. Furthermore, treatment of leukemia cells with combretastatins induced a bottleneck at the G2/M checkpoint, resulting in increased proportion of leukemia cells at the S phase (39.1% (CA1) and 38.4% (CA4) vs. 29%, p < .001) and at the G2/M phase (48.2% (CA1) and 45.9% (CA4) vs. 10.4%, p < .0005), suggesting that combretastatins sensitize AML cells against cell-cycle cytotoxic agents. Finally, human AML xenograft experiments showed a complete clearance of AML cells in the bone marrow after combination treatment with low dose CA1P (OXi4503) and Ara-C or azacitidine; whereas Ara-C or azacitidine alone showed limited effect on AML engraftment in the bone marrow.Conclusion: BMECs protect AML cells from conventional cytotoxic chemotherapy partly through adhesion factors. However, this protection can be removed by pretreating the leukemia microenvironment with vascular disrupting combretastatins. Combination combretastatin + cytotoxic chemotherapy sensitizes AML cells and leads to improved leukemia regression. Results of this study support a phase IB study of OXi4503 and chemotherapy in patients with AML and advanced myelodysplastic syndrome. DisclosuresNo relevant conflicts of interest to declare.

Cell confluency modulates cortical stiffness of human bone marrow endothelial cells (696.3)

Little is known about mechanical properties of human bone marrow endothelial cells (HBMEC) as culture conditions change. Here, we used atomic force microscopy (AFM) to test the hypothesis that the degree of confluency modifies HBMEC stiffness. Individual cells were compared in non‐confluent and confluent phases. Two HBMEC lines HBME‐1 and HBMEC‐60 immortalized by different methods were cultured until reaching a desired growth phase. Primary HBMEC were used as a reference. Cell stiffness was calculated as Young’s Moduli using a modified Hertz model from AFM cell‐surface force curves. Actin expression was analyzed by immunofluorescence and western blotting (WB). Young’s Moduli were significantly increased in confluent vs non‐confluent HBME‐1 (6.79±2.58 vs 4.73±1.78 kPa, p&lt;0.01) and HBMEC‐60 (4.25±1.14 vs 2.03±1.09 kPa, p&lt;0.01) cells. Non‐confluent HBME‐1 exhibited similar stiffness to the primary HBME (5.49±2.06 kPa). Topographical images revealed denser networks of actin stress fibers in confluent cells. F‐actin labeling confirmed these results. WB data indicated elevated pan‐actin levels in confluent HBMEC. Thus, HBMEC stiffness is influenced by cell confluency in culture and enhanced stiffness is associated with increased actin expression, a major cytoskeletal component contributing to cell mechanical properties.Grant Funding Source: Supported by NIH Grants HL095486 (GAM), CA160461 (VVG), VA BLR&amp;D Service Award 1I01BX000609 (VVG)

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

Bradykinin stimulation of nitric oxide production is not sufficient for gamma-globin induction.

Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases gamma-globin and fetal hemoglobin levels in human erythroid progenitors. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase gamma-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 microM up to 30 minutes).This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/beta-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase gamma-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce gamma/beta globin ratio in erythroid progenitors in co-cultures with HUVEC. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce gamma-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of gamma-globin gene expression.

Comparison Of Autologous Microparticles and Fucosyltransferase-7 For Adhesion Improvement Of Hematopoietic Stem Cells To Bone Marrow Endothelial Cells In a Microfluidic Flow Chamber

The improvement of graft function and time to engraftment might help to reduce infection-related mortality in stem cell transplantation (SCT). While the concept of stem cells fucosylation for accelerated engraftment has already reached clinical study phase (for cord blood transplantation; NCT01471067), own previous work has shown an association between engraftment time and circulating microparticles bearing P-Selectin and P-Selectin glycoprotein ligand 1 (PSGL-1). PSGL-1 contains the sialyl Lewis x (CD15s) antigen that requires fucosylation for optimal binding of P- and E-Selectin on endothelial cells. We therefore hypothesized that addition of microparticles (MP) might enhance adhesion of human stem cells (HSC) to bone marrow endothelial cells and that MP might have synergistic effects in combination with stem cell fucosylation. HSC were obtained from apheresis products of allogeneic donors, purified by Ficoll and magnetic bead separation for CD34, stained with calcein AM and perfused through an automated microfluidic flow chamber (Bioflux 200, Fluxionbio, USA) covered with a confluent layer of an immortalized human bone marrow endothelial cell line (HBMEC). Photos (and videos) were taken using a fluorescence microscope at start, 5 min and 10 min and analyzed for adherent HSC across the whole chamber (about 1.5 sqmm) using ImageJ software. Autologous MP were generated by addition of calcimycin to apheresis and isolation of MP by centrifugation. For control experiments, one part of the MP solution was passed through a 0,2µm-filter to remove MP. MP concentration (mean: 1362/µl) was assessed by detection of Annexin V binding in flow cytometry, using TrucountBeads® for quantification. Fucosylation was performed by 1h incubation of isolated CD34+ stem cells with GDP-fucose and fucosyltransferase 7 (FUT7). Successful fucosylation was controlled by CD15s staining of HSC in flow cytometry. Results of seven experiments (in duplicate) demonstrated the highest number of adherent HSC in the MPpositiv/FUT7negativ preparation (median: 32 HSC/sqmm; range: 15-78), followed by MPpositiv/FUT7positiv (30 HSC/sqmm; range: 16-38), MPnegativ/FUT7positiv (median: 25/sqmm; range: 11-27) and MPnegativ/FUT7negativ (20 HSC/sqmm; range: 0-22). Comparison of the MPpositiv/FUT7negativ and MPnegativ/FUT7negativ as well as the MPpositiv/FUT7positiv and MPpositiv/FUT7negativ preparations showed statistically significant differences in Wilcoxon rank test (p<.05) while comparison of MPpositiv/FUT7positiv vs. MPnegativ/FUT7positiv and MPnegativ/FUT7positiv vs. MPnegativ/FUT7negativ preparations did not. In summary, these results demonstrate that MP can improve HSC adhesion to bone marrow endothelial cells similar to fucosylation. The effect of fucosylation on HSC adhesion appears to be mediated by MP. However, there is not a synergistic effect between MP and fucosylation. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Endothelial Cells Protect Acute Myeloid Leukemia From Chemotherapy By Direct Contact: The BCAM/Laminin/VLA5 Axis As a Potential Therapeutic Target

Causes of refractory acute myeloid leukemia (AML) are unknown, but may be related to the bone marrow (BM) vascular network given the close relationship between hematopoiesis and the vasculature. We hypothesized that endothelial cells (ECs) provide a protective advantage to AML cells. To test this hypothesis, we first cultured human AML cells with and without primary bone marrow endothelial cells (BMECs). Co-cultures of AML cells and BMECs were then exposed to increasing doses of cytarabine chemotherapy. There was a 2-fold decrease in leukemia cell death of AML cells when adhered to BMECs compared to non-adhered (30% vs. 60%, P < 0.0001). Even irradiated BMECs protected AML cells from cytarabine chemotherapy. To identify adhesion molecules mediating this protective effect, we analyzed cell membranes and supernatants of the cytarabine-treated co-cultures using protein microarrays. After cytarabine exposure, the Lutheran blood group glycoprotein basal cell adhesion molecule (BCAM) was upregulated in both human AML cells and BMECs. Prior work has shown BCAM as a receptor for Laminin and VLA5. As AML cells are known to express VLA5, we hypothesized that blocking BCAM may represent a novel therapeutic strategy. Blocking BCAM with neutralizing antibodies resulted in a 75% increase in non-adherent AML cells. Together, these in vitro results support the concept that ECs may be a protective reservoir for AML cells, at a minimum by means of adhesion molecules. BCAM represents a viable target. Because of the complex nature of the leukemia microenvironment, we sought to test this concept in vivo. Prior intravital efforts have focused on calvaria bone, which may over-represent the endosteal niche and under-represent the vascular niche due to the very close approximation of bone surfaces. Therefore, we created an intravital animal model of human AML to track single AML cells in the bone marrow of mouse long bones. In brief, we irradiated NOD/scid/IL2Rγnull (NSG) mice, drilled a window on the tibia surface, xenotransplanted fluorescently tagged human AML cells via IV injection, and then analyzed the tibias by fluorescent microscopy for the presence of AML cells at various time points after transplant. Initially the AML cells homed to endosteal surfaces of the bone marrow as early as one day after transplant. Over time, the AML cells lining the endosteum remained as single cells or very small clusters of a few cells. However, in the central marrow region the AML cells proliferated into massive clusters around blood vessels. Ongoing experiments are being performed to determine the disruption and resurgence of AML cells after chemotherapy and blockade of adhesion molecules. In sum, ECs protect human AML cells from chemotherapy by direct contact and the BCAM/Laminin/VLA5 axis may be a therapeutic target. Using our unique intravital imaging model of bone marrow in long bones, the endosteal niche appears to be the first site of homing and engraftment while the vascular niche appears to be the site for leukemia proliferation/progression. Disclosures:No relevant conflicts of interest to declare.

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7

BackgroundCD7 expression is found on ~30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7+) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation. MethodsKG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7+, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCβII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA. ResultsSECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCβII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression. ConclusionThese findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis. General SignificanceThis work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.

Whole genome expression profiling and screening for differentially expressed cytokine genes in human bone marrow endothelial cells treated with humoral inhibitors in liver cirrhosis

Bone marrow endothelial cells (BMECs) are important components of the hematopoietic microenvironment in bone marrow, and they can secrete several types of cytokines to regulate the functions of hematopoietic stem/progenitor cells. To date, it is unknown whether BMECs undergo functional changes and lead to hematopoietic abnormalities in cases of liver cirrhosis (LC). In the present study, whole genome microarray analysis was carried out to detect differentially expressed genes in human BMECs treated for 48 h with medium supplemented with 20% pooled sera from 26 patients with LC or 10 healthy volunteers as the control group. A total of 1,106 upregulated genes and 766 downregulated genes were identified. In Gene Ontology analysis, the most significant categories of genes were revealed. A large number of the upregulated genes were involved in processes, such as cell-cell adhesion, apoptosis and cellular response to stimuli and the downregulated genes were involved in the negative regulation of secretion, angiogenesis, blood vessel development and cell growth. Pathway analysis revealed that the upregulated genes were either cell adhesion molecules or parts of the apoptotic signaling pathway and the downregulated genes were involved in the Wnt signaling pathway and MAPK signaling pathway. These were the pathways with the highest enrichment scores. The results of apoptosis assays revealed that the humoral inhibitors in the sera of patients with LC induced the apoptosis of BMECs, which confirmed the accuracy of bioinformatic analysis. Moreover, we screened and verified 21 differentially expressed cytokine genes [transforming growth factor (TGF)B1, tumor necrosis factor (TNF)B, TNF receptor superfamily, member 11b (TNFRSF11B), TNF (ligand) superfamily, member 13b (TNFSF13B), interleukin (IL)1A, IL6, IL11, IL17C, IL24, family with sequence similarity 3, member B (FAM3B), Fas ligand (FASLG), matrix metallopeptidase (MMP)3, MMP15, vitronectin (VTN), insulin-like growth factor 1 (IGF1), fibroblast growth factor 22 (FGF22), slit homolog 2 (Drosophila) (SLIT2), thrombospondin (THBS)2, THBS3, chemokine (C-C motif) ligand 28 (CCL28) and macrophage stimulating 1 (MST1)] from 97 cytokine genes in BMECs treated with serum from patients with LC. The results from our study demonstrate that the humoral inhibitors in the sera of patients with LC induce the dysfunction and abnormal cytokine secretion by BMECs, which may be a novel mechanism responsible for hematological abnormalities in patients with LC.