Modulation of angiogenesis during adipose tissue development in murine models of obesity.

peer reviewedDevelopment of vasculature and mRNA expression of 17 pro- or antiangiogenic factors were studied during adipose tissue development in nutritionally induced or genetically determined murine obesity models. Subcutaneous (SC) and gonadal (GON) fat pads were harvested from male C57Bl/6 mice kept on standard chow [standard fat diet (SFD)] or on high-fat diet for 0-15 wk and from male ob/ob mice kept on SFD. Ob/ob mice and C57Bl/6 mice on high-fat diet had significantly larger SC and GON fat pads, accompanied by significantly higher blood content, increased total blood vessel volume, and higher number of proliferating cells. mRNA and protein levels of angiopoietin (Ang)-1 were down-regulated, whereas those of thrombospondin-1 were up-regulated in developing adipose tissue in both obesity models. Ang-1 mRNA levels correlated negatively with adipose tissue weight in the early phase of nutritionally induced obesity as well as in genetically determined obesity. Placental growth factor and Ang-2 expression were increased in SC adipose tissue of ob/ob mice, and thrombospondin-2 was increased in both their SC and GON fat pads. mRNA levels of vascular endothelial growth factor (VEGF)-A isoforms VEGF-B, VEGF-C, VEGF receptor-1, -2, and -3, and neuropilin-1 were not markedly modulated by obesity. This modulation of angiogenic factors during development of adipose tissue supports their important functional role in obesity

Stromal integrin α11 regulates PDGFR-β signaling and promotes breast cancer progression

Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors, such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRβ–positive CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11 deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRβ was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRβ expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using 5 CAF subpopulations (1 murine, 4 human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, the proinvasive activity of integrin α11 relies on its ability to interact with PDGFRβ in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a proinvasive matricellular protein. Pharmacological inhibition of PDGFRβ and JNK impaired tumor cell invasion induced by integrin α11+ CAFs. Collectively, our study uncovers an integrin α11+ subset of protumoral CAFs that exploits the PDGFRβ/JNK signaling axis to promote tumor invasiveness in BC.publishedVersio

A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

<p>Abstract</p> <p>Background</p> <p>The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of <it>in-vivo </it>invasion assays, there is need for quantitative <it>in-vitro </it>invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled.</p> <p>Methods</p> <p>We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively.</p> <p>Results</p> <p>The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2) is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells.</p> <p>Conclusion</p> <p>Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on tumor cell invasiveness.</p

Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion

Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes: increase of lipid catabolism and reduction of adipokine expression

Aims/hypothesis: The thiazolidinedione (TZD) rosiglitazone is a peroxisome proliferator-activated receptor-¿ agonist that induces adipocyte differentiation and, hence, lipid accumulation. This is in apparent contrast to the long-term glucose-lowering, insulin-sensitising effect of rosiglitazone. We tested whether the action of rosiglitazone involves specific effects on mature adipocytes, which are different from those on preadipocytes. Materials and methods: Differentiated mature 3T3-L1 adipocytes were used as an in vitro model. Transcriptomics, proteomics and assays of metabolism were applied to assess the effect of rosiglitazone in different insulin and glucose conditions. Results: Rosiglitazone does not induce an increase, but rather a decrease in the lipid content of mature adipocytes. Analysis of transcriptome data, confirmed by quantitative RT-PCR and measurements of lipolysis, indicates that an altered energy metabolism may underlie this change. The pathway analysis shows a consistent picture dominated by lipid catabolism. In addition, we confirmed at both mRNA level and protein level that rosiglitazone represses adipokine expression and production, except for genes encoding adiponectin and apolipoprotein E. Moreover, transcriptome changes indicate that a general repression of genes encoding secreted proteins occurs. Conclusions/ interpretation: Our findings suggest that the change of adiposity as seen in vivo reflects a shift in balance between the different effects of TZDs on preadipocytes and on mature adipocytes, while the changes in circulating adipokine levels primarily result from an effect on mature adipocyte

Влияние фосфатных связующих на физико-механические свойства периклазохромитовых огнеупоров

У данній статті наведено та порівняно фізико-механічні властивості периклазо-хромітових матеріалів в залежності від різних типів фосфатних зв’язуючих та введення різних домішок. Визначено, що найбільш раціональним є введення триполіфосфату натрію.In given clause are resulted and the physycal-mechanical properties periclase-cgromite of materials are compared depending on different of types phosphate binding and introduction of the various additives. Is determined, that most rational is the introduction treepolyphosphate sodume

Quantifying the Proteolytic Release of Extracellular Matrix-Sequestered VEGF with a Computational Model

BACKGROUND: VEGF proteolysis by plasmin or matrix metalloproteinases (MMPs) is believed to play an important role in regulating vascular patterning in vivo by releasing VEGF from the extracellular matrix (ECM). However, a quantitative understanding of the kinetics of VEGF cleavage and the efficiency of cell-mediated VEGF release is currently lacking. To address these uncertainties, we develop a molecular-detailed quantitative model of VEGF proteolysis, used here in the context of an endothelial sprout. METHODOLOGY AND FINDINGS: To study a cell's ability to cleave VEGF, the model captures MMP secretion, VEGF-ECM binding, VEGF proteolysis from VEGF165 to VEGF114 (the expected MMP cleavage product of VEGF165) and VEGF receptor-mediated recapture. Using experimental data, we estimated the effective bimolecular rate constant of VEGF165 cleavage by plasmin to be 328 M(-1) s(-1) at 25 degrees C, which is relatively slow compared to typical MMP-ECM proteolysis reactions. While previous studies have implicated cellular proteolysis in growth factor processing, we show that single cells do not individually have the capacity to cleave VEGF to any appreciable extent (less than 0.1% conversion). In addition, we find that a tip cell's receptor system will not efficiently recapture the cleaved VEGF due to an inability of cleaved VEGF to associate with Neuropilin-1. CONCLUSIONS: Overall, VEGF165 cleavage in vivo is likely to be mediated by the combined effect of numerous cells, instead of behaving in a single-cell-directed, autocrine manner. We show that heparan sulfate proteoglycans (HSPGs) potentiate VEGF cleavage by increasing the VEGF clearance time in tissues. In addition, we find that the VEGF-HSPG complex is more sensitive to proteases than is soluble VEGF, which may imply its potential relevance in receptor signaling. Finally, according to our calculations, experimentally measured soluble protease levels are approximately two orders of magnitude lower than that needed to reconcile levels of VEGF cleavage seen in pathological situations

Role of Matrix Metalloproteinases and Therapeutic Benefits of Their Inhibition in Spinal Cord Injury

This review will focus on matrix metalloproteinases (MMPs) and their inhibitors in the context of spinal cord injury (SCI). MMPs have a specific cellular and temporal pattern of expression in the injured spinal cord. Here we consider their diverse functions in the acutely injured cord and during wound healing. Excessive activity of MMPs, and in particular gelatinase B (MMP-9), in the acutely injured cord contributes to disruption of the blood-spinal cord barrier, and the influx of leukocytes into the injured cord, as well as apoptosis. MMP-9 and MMP-2 regulate inflammation and neuropathic pain after peripheral nerve injury and may contribute to SCI-induced pain. Early pharmacologic inhibition of MMPs or the gelatinases (MMP-2 and MMP-9) results in an improvement in long-term neurological recovery and is associated with reduced glial scarring and neuropathic pain. During wound healing, gelatinase A (MMP-2) plays a critical role in limiting the formation of an inhibitory glial scar, and mice that are genetically deficient in this protease showed impaired recovery. Together, these findings illustrate complex, temporally distinct roles of MMPs in SCIs. As early gelatinase activity is detrimental, there is an emerging interest in developing gelatinase-targeted therapeutics that would be specifically tailored to the acute injured spinal cord. Thus, we focus this review on the development of selective gelatinase inhibitors