Delfim Duarte

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells.

Dynamics of Intracellular Clathrin/AP1- and Clathrin/AP3-Containing Carriers

Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, σ adaptor subunits, or dynamin2) with a spatial precision of up to ~30 nm and a temporal resolution of ~1 s. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that although dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant-negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers.

Angiogenesis and Inflammation Signaling Are Targets of Beer Polyphenols on Vascular Cells

Emerging evidence indicates that chronic inflammation and oxidative stress cluster together with angiogenic imbalance in a wide range of pathologies. In general, natural polyphenols present health‐protective properties, which are likely attributed to their effect on oxidative stress and inflammation. Hops used in beer production are a source of polyphenols such as xanthohumol (XN), and its metabolites isoxanthohumol (IXN) and phytoestrogen 8‐prenylnaringenin (8PN). Our study aimed to evaluate XN, IXN, and 8PN effects on angiogenesis and inflammation processes. Opposite in vitro effects were observed between 8PN, stimulating endothelial and smooth muscle cell (SMC) growth, motility, invasion and capillary‐like structures formation, and XN and IXN, which inhibited them. Mouse matrigel plug and rat skin wound‐healing assays confirmed that XN and IXN treatments reduced vessel number as well as serum macrophage enzymatic activity, whereas 8PN increased blood vessels formation in both assays and enzyme activity in the wound‐healing assay. A similar profile was found for serum inflammatory interleukin‐1β quantification, in the wound‐healing assay. Our data indicate that whereas 8PN stimulates angiogenesis, XN and IXN manifested anti‐angiogenic and anti‐inflammatory effects in identical conditions. These findings suggest that the effects observed for individual compounds on vascular wall cells must be carefully taken into account, as these polyphenols are metabolized after in vivo administration. The modulation of SMC proliferation and migration is also of special relevance, given the role of these cells in many pathological conditions. Furthermore, these results may provide clues for developing useful therapeutic agents against inflammation‐ and angiogenesis‐associated pathologies. J. Cell. Biochem. 111: 1270–1279, 2010.

Xanthohumol Modulates Inflammation, Oxidative Stress, and Angiogenesis in Type 1 Diabetic Rat Skin Wound Healing

Type 1 diabetes mellitus is responsible for metabolic dysfunction, accompanied by chronic inflammation, oxidative stress, and endothelium dysfunction, and is often associated with impaired wound healing. Phenol-rich food improves vascular function, contributing to diabetes prevention. This study has evaluated the effect of phenol-rich beverage consumption in diabetic rats on wound healing, through angiogenesis, inflammation, and oxidative stress modulation. A wound-healing assay was performed in streptozotocin-induced diabetic Wistar rats drinking water, 5% ethanol, and stout beer with and without 10 mg/L xanthohumol (1), for a five-week period. Wounded skin microvessel density was reduced to normal values upon consumption of 1 in diabetic rats, being accompanied by decreased serum VEGF-A and inflammatory markers (IL-1β, NO, N-acetylglucosaminidase). Systemic glutathione and kidney and liver H2O2, 3-nitrotyrosine, and protein carbonylation also decreased to healthy levels after treatment with 1, implying an improvement in oxidative stress status. These findings suggest that consumption of xanthohumol (1) by diabetic animals consistently decreases inflammation and oxidative stress, allowing neovascularization control and improving diabetic wound healing.

Different effects of catechin on angiogenesis and inflammation depending on VEGF levels

Although physiological and pathological angiogenesis develop through similar processes, during pathological angiogenesis, proangiogenic factors are exacerbated. Polyphenols have been considered therapeutic tools for conditions exhibiting enhanced angiogenesis. However, the possibility that these compounds may also prevent vascularization in physiological situations is a major drawback for their use. The purpose of the current study was to investigate the effects of 0.1-100 μM catechin on endothelial cells (EC) and vascular smooth muscle cells (VSMC) regarding angiogenic and inflammatory processes. Catechin modulation of angiogenesis and inflammation was also evaluated in vivo using different models of angiogenesis: one physiological (skin wound-healing assay) and another one resembling pathological angiogenesis, exhibiting higher vascular endothelial growth factor (VEGF)-A stimulation (Matrigel plug assay). The in vitro results showed that 100 μM catechin increased viability (to 165.58% and to 165.34%) and decreased apoptosis (53.45% and 92.65%) and proliferation (33.19% and 23.36%) of EC and VSMC, respectively. Catechin affected migration and invasion, tending to increase both in EC and decreasing them in VSMC; however, it did not change sprouting angiogenesis. Nevertheless, catechin diminished in vitro inflammatory modulators such as tumor necrosis factor α (58.66% for human umbilical vein endothelial cells and 85.46% for human aortic smooth muscle cells) and nuclear factor kappa-B (38.43% for VSMC). The in vivo results demonstrated that catechin did not change angiogenesis and inflammation in skin wound-healing model and substantially decreased these processes in Matrigel plug assay. Altogether, the current study showed that catechin has different effects in angiogenesis and inflammation depending on VEGF-A levels. The absence of adverse effects in mature vasculature favors catechin potential use against pathological situations where angiogenesis is stimulated.

Increased circulating platelet microparticles as a potential biomarker in asthma.

Endothelial (EMPs) and platelet microparticles (PMPs) have been studied as biomarkers in several inflammatory diseases and as central players in intercellular communication.

Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML

Summary Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML.

Automated Identification and Localization of Hematopoietic Stem Cells in 3D Intravital Microscopy Data

Summary Measuring three-dimensional (3D) localization of hematopoietic stem cells (HSCs) within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components.

Xanthohumol‐supplemented beer modulates angiogenesis and inflammation in a skin wound healing model. Involvement of local adipocytes

Angiogenesis and inflammation are two intermingled processes that play a role in wound healing. Nevertheless, whenever exacerbated, these processes result in nonhealing wounds. Xanthohumol (XN), a beer‐derived polyphenol, inhibits these processes in many physiopathological situations. This study aimed at examining whether XN ingestion affects wound healing. Wistar rats drinking water, 5% ethanol, stout beer (SB) or stout beer supplemented with 10 mg/L XN (Suppl SB) for 4 weeks, were subjected to a 1.5 cm full skin‐thickness longitudinal incision, and further maintained under the same beverage conditions for another week. No differences in beverage consumption or body weight were found throughout the study but food intake decreased in every group relative to controls. Consumption of Suppl SB resulted in decreased serum VEGF levels (18.42%), N‐acetylglucosaminidase activity (27.77%), IL1β concentration (9.07%), and NO released (77.06%), accompanied by a reduced redox state as observed by increased GSH/GSSG ratio (to 198.80%). Also, the number of blood vessels within the wound granulation tissue seems to reduce in animals drinking Suppl SB (23.08%). Interestingly, SB and primarily Suppl SB showed a tendency to increase adipocyte number (to 194.26% and 156.68%, respectively) and reduce adipocyte size (4.60% and 24.64%, respectively) within the granuloma. Liver function and metabolism did not change among the animal groups as analyzed by plasma biochemical parameters, indicating no beverage toxicity. This study shows that XN intake in its natural beer context reduced inflammation, oxidative stress, and angiogenesis, ameliorating the wound healing process, suggesting that this polyphenol may exert beneficial effect as a nutritional supplement. J. Cell. Biochem. 113: 100–109, 2012.

Isoxanthohumol modulates angiogenesis and inflammation via vascular endothelial growth factor receptor, tumor necrosis factor alpha and nuclear factor kappa B pathways

Angiogenesis and inflammation are becoming distinguished players in the pathogenesis of many heterogeneous diseases, such as diabetes, cardiovascular disease, and cancer. Therefore, it is crucial to study new compounds that are able to modulate these events. Isoxanthohumol (IXN) is a polyphenol with antioxidant, anti-inflammatory, and antiangiogenic properties. The aim of this study was to evaluate the effects of IXN on blood vessel proliferation and maturation and describe underlying molecular mechanisms in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Angiogenic profile of IXN was analyzed by retinal angiogenesis at different time points. IXN modulation of angiogenic and inflammatory signaling pathways was evaluated by Western blotting on EC and VSMC cultures. IXN inhibited by 20% sprouting angiogenesis and decreased vascular coverage by mural cells up to 39%. IXN of 10 µM also decreased inflammatory signals, namely tumor necrosis factor alpha (TNF-α) (26 and 40%) and factor nuclear kappa B (24 and 42%) in human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs). Angiogenic regulators, including vascular endothelial growth factor receptor 2 (HUVEC, 55%), angiopoietins 1 (HUVEC, 39%; HASMC, 35%), angiopoietin 2 (HUVEC, 38%), and Tie2 (HUVEC, 56%) were also inhibited by 10 µM of IXN treatments. Akt activation was reduced by 47% in HUVEC-treated cells and Erk activation was also reduced by 52 and 69% upon IXN treatment of HUVEC and HASMC. IXN seems to regulate in vivo vascular proliferation and stabilization and the EC-VSMC-inflammatory crosstalk, leaving this molecule as an interesting nutritional player for angiogenesis and inflammation-related diseases.