an-efficient-reproducible-protocol-for-distraction-osteogenesis-rat

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Aix Marseille Univ

APHM

Pathological levels of homocysteine induce a metalloproteinase-dependent degradation of the elastic structures in arterial wall. This elastolytic process is preferentially localized toward the internal elastic laminae and in the first layers of the media, suggesting endothelium could participate in extracellular matrix degradation induced by homocysteine. Therefore, we studied the effects of homocysteine on proteolytic potential of endothelial cells. Human umbilical vein endothelial cells were cultured with concentrations of homocysteine matching human physiological (10 microM) and pathological (50, 100, and 250 microM) plasma homocysteine levels. Pathological levels of homocysteine increased the secretion of elastolytic metalloproteinase-2 and -9, but not of metalloproteinase-3 and -7. Homocysteine also increased the expression of human tissue kallikrein, a potential activator of matrix metalloproteinase-2 and -9, while the expression of urokinase plasminogen activator was not altered. These results suggest vascular endothelial cells could participate in the subendothelial degradation of the arterial elastic structures occurring in hyperhomocysteinemia.

Homocysteine modulates the proteolytic potential of human vascular endothelial cells.

Endothelial progenitor cells (EPC) display a unique ability to repair vascular injury and promote neovascularization although the underlying molecular mechanisms remain poorly understood. Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play a critical role in cell migration and angiogenesis by facilitating proteolysis of extracellular matrix. The aim of the present study was to characterize the uPA/uPAR-dependent proteolytic potential of EPC outgrown from human umbilical cord blood and to analyze its contribution to their angiogenic properties in vitro. Cells derived from EPC (EPDC), presenting typical features of late outgrowth endothelial cells, were compared to mature endothelial cells, represented by human umbilical vein endothelial cells (HUVEC). Using quantitative flow cytometry, enzyme-linked immunosorbent assays and zymography, we demonstrated that EPDC displayed higher levels of uPA and uPAR. In conditioned culture media, uPA-dependent proteolytic activity was also found to be significantly increased in EPDC. This activity was paralleled by a higher secretion of pro-metalloproteinase-2 (pro-MMP-2). Inhibition of EPDC-associated uPA by monoclonal antibodies that block either uPA activity or receptor binding, significantly reduced proliferation, migration and capillary like tube formation. Moreover, tumor necrosis factor-alpha and vascular endothelial growth factor, known to be locally secreted in ischemic areas, further increased the proteolytic potential of EPDC by up-regulating uPA and uPAR expression respectively. The EPDC response to these factors was found to be more pronounced than that of HUVEC. In conclusion, these findings indicated that EPDC are characterized by high intrinsic uPA/uPAR-dependent proteolytic potential that could contribute to their invasive and angiogenic behaviour.

High urokinase expression contributes to the angiogenic properties of endothelial cells derived from circulating progenitors.

The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cell-derived microparticles participate in the dissemination of biological activities. However, their capacity to promote plasmin generation has not been documented. In this study, we show that endothelial microparticles (EMPs) from tumor necrosis factor alpha (TNFalpha)-stimulated endothelial cells served as a surface for the generation of plasmin. The generation of plasmin involved expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) at the surface of EMPs and was further increased by their ability to bind exogenous uPA on uPAR. Plasminogen was activated at the surface of EMPs in a dose-dependent, saturable, and specific manner as indicated by the inhibition of plasmin formation by epsilon-amino-caproic acid (epsilon-ACA) and carboxypeptidase B. EMP-induced plasmin generation affects tube formation mediated by endothelial progenitor cells. However, low amounts of EMPs increased tube formation, whereas higher concentrations inhibited it. Prevention of these effects by inhibitors of either uPA or plasmin underscore the key role of EMP-induced plasmin generation. In conclusion, we demonstrated that EMPs act as vectors supporting efficient plasmin generation and dissemination, a new pathway in the regulation of endothelial proteolytic activities with potential involvement in inflammation, angiogenesis, and atherosclerosis.

Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro.

The thermal and dielectric properties of the elastin network were investigated in arteries cultured with physiological and pathological concentrations of homocysteine, an aminoacid responsible of histological impairments in human arteries. The physical structure of this amorphous protein was investigated by differential scanning calorimetry (DSC). To explore the molecular dynamics of the elastin network in the nanometer range, we used thermally stimulated currents (TSC), a dielectric technique running at low frequency, and measuring the dipolar reorientations in proteins subjected to a static electrical field. Combining DSC and TSC experiments reveals the molecular mobility of the proteins, both in the glassy state and in the liquid state. Significant differences are evidenced in the physical structure and relaxation behavior of elastin network in cultured arteries (physiological and pathological concentrations of homocysteine) and discussed.

Changes in the physical structure and chain dynamics of elastin network in homocysteine-cultured arteries.

Pathological levels of homocysteine induce a dramatic degradation of arterial elastic structures. This severe metalloproteinase-dependant elastolysis affects elastic structures all over the media suggesting that smooth muscle cells (SMC) may participate to this process induced by homocysteine. Therefore, we investigated the effect of physiological (10 microM) and pathological (50, 100, and 500 microM) concentrations of homocysteine on the metalloproteinase-dependant proteolytic potential of human arterial SMC in culture. Pathological levels of homocysteine increased concomitantly the secretion of latent MMP-2 and TIMP-2 while the secretion of other elastolytic matrix metalloproteinases (MMPs) and expression of MT1-MMP were not altered. The increased secretion of latent MMP-2 induced by homocysteine was associated with an increased production of reactive oxygen species (ROS). Moreover, the increased secretion of latent MMP-2 induced by homocysteine was inhibited by antioxidant superoxide dismutase alone or in combination with catalase. These results suggest that SMC could participate, through an oxidative stress dependant secretion of elastolytic MMP-2, to the metalloproteinase-dependant degradation of arterial elastic structures induced by homocysteine.

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism.

CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases.

Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia.

CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined.

CD146 short isoform increases the proangiogenic potential of endothelial progenitor cells in vitro and in vivo.

Low birth weight (LBW) is associated with increased risk of cardiovascular diseases at adulthood. Nevertheless, the impact of LBW on the endothelium is not clearly established. We investigate whether LBW alters the angiogenic properties of cord blood endothelial colony forming cells (LBW-ECFCs) in 25 preterm neonates compared with 25 term neonates (CT-ECFCs). We observed that LBW decreased the number of colonies formed by ECFCs and delayed the time of appearance of their clonal progeny. LBW dramatically reduced LBW-ECFC capacity to form sprouts and tubes, to migrate and to proliferate in vitro. The angiogenic defect of LBW-ECFCs was confirmed in vivo by their inability to form robust capillary networks in Matrigel plugs injected in nu/nu mice. Gene profile analysis of LBW-ECFCs demonstrated an increased expression of antiangiogenic genes. Among them, thrombospondin 1 (THBS1) was highly expressed at RNA and protein levels in LBW-ECFCs. Silencing THBS1 restored the angiogenic properties of LBW-ECFCs by increasing AKT phosphorylation. The imbalance toward an angiostatic state provide a mechanistic link between LBW and the impaired angiogenic properties of ECFCs and allows the identification of THBS1 as a novel player in LBW-ECFC defect, opening new perspectives for novel deprogramming agents.

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants.

Ultrasonic backscatter coefficient (BSC) measurements were performed on K562 cell pellet biophantoms with cell concentrations ranging from 0.006 to 0.30 in the 10-42 MHz frequency bandwidth. Three scattering models, namely, the fluid-filled sphere model (FFSM), the particle model (PM), and the structure factor model (SFM), were compared for modeling the scattering from an ensemble of concentrated cells. A parameter estimation procedure was developed in order to estimate the scatterer size and relative impedance contrast that could explain the measured BSCs from all the studied cell concentrations. This procedure was applied to the BSC data from K562 cell pellet biophantoms in the 10-42 MHz frequency bandwidth and to the BSC data from Chinese hamster ovary cell pellet biophantoms in the 26-105 MHz frequency bandwidth given in Han, Abuhabsah, Blue, Sarwate, and O'Brien [J. Acoust. Soc. Am. 130, 4139-4147 (2011)]. The data fitting quality and the scatterer size estimates show that the SFM was more suitable than the PM and the FFSM for modeling the responses from concentrated cell pellet biophantoms.

Article	Year
Homocysteine modulates the proteolytic potential of human vascular endothelial cells. Biochemical and biophysical research communications\| PubMed ID: 15003526	2004
High urokinase expression contributes to the angiogenic properties of endothelial cells derived from circulating progenitors. Thrombosis and haemostasis\| PubMed ID: 16601839	2006
Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro. Blood\| PubMed ID: 17606760	2007
Changes in the physical structure and chain dynamics of elastin network in homocysteine-cultured arteries. Journal of biomedical materials research. Part A\| PubMed ID: 19609876	2010
Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism. Molecular and cellular biochemistry\| PubMed ID: 19787299	2010
Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia. Blood\| PubMed ID: 20185588	2010
CD146 short isoform increases the proangiogenic potential of endothelial progenitor cells in vitro and in vivo. Circulation research\| PubMed ID: 20448216	2010
A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants. Blood\| PubMed ID: 21659549	2011
Structure factor model for understanding the measured backscatter coefficients from concentrated cell pellet biophantoms. The Journal of the Acoustical Society of America\| PubMed ID: 24916409	2014

Edouard Lamy

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