Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo.

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Interleukin-7-Dependent Production of RANTES That Correlates with Human Immunodeficiency Virus Disease Progression

Journal of Virology ◽

10.1128/jvi.77.7.4389-4395.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4389-4395 ◽

Cited By ~ 16

Author(s):

Anuska Llano ◽

Jordi Barretina ◽

Arantxa Gutiérrez ◽

Bonaventura Clotet ◽

José A. Esté

Keyword(s):

Human Immunodeficiency Virus ◽

Disease Progression ◽

Mononuclear Cells ◽

Ex Vivo ◽

Virus Disease ◽

Chemokine Production ◽

Interleukin 7 ◽

Cell Counts ◽

Immunodeficiency Virus

ABSTRACT There is a relationship between CD4-T-cell number and circulating interleukin 7 (IL-7) levels in human immunodeficiency virus (HIV)-positive individuals. Here, we show that IL-7 induced a dose-dependent production of CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) in peripheral blood mononuclear cells (PBMC), ex vivo tonsil lymphoid tissue of HIV− individuals, and PBMC from HIV+ individuals, suggesting that IL-7 may regulate β-chemokine production in vivo. In a cross-sectional study of HIV+ individuals (n = 130), a weak but significant correlation between IL-7 and RANTES was noted (r = 0.379; P < 0.001). Remarkably, the correlation between IL-7 and RANTES increased to an r value of 0.798 (P < 0.001) if individuals with low CD4 cell counts (<200 cells/μl) were excluded from the analysis. Our results suggest that there is a relationship between IL-7 and the production of RANTES both in vitro and in vivo that is lost in immune-compromised patients (CD4 count of <200 cells/μl) but that could be restored by antiretroviral therapy. Unlike the case for IL-7, high levels of RANTES suggest an intermediate stage of HIV disease progression.

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My D88-Dependent Interplay Between Myeloid and Endothelial Cells in the Initiation and Progression of Obesity-Associated Inflammatory Diseases

Blood ◽

10.1182/blood.v128.22.sci-44.sci-44 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-44-SCI-44

Author(s):

Xiaoxia Li

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

Adipose Tissue ◽

Systemic Inflammation ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Inflammatory Diseases ◽

Critical Role ◽

Low Grade

Abstract Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

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Influence of genetic background on ex vivo and in vivo cardiac function in several commonly used inbred mouse strains

Physiological Genomics ◽

10.1152/physiolgenomics.00071.2010 ◽

2010 ◽

Vol 42A (2) ◽

pp. 103-113 ◽

Cited By ~ 54

Author(s):

Matthew S. Barnabei ◽

Nathan J. Palpant ◽

Joseph M. Metzger

Keyword(s):

Cardiac Function ◽

Genetic Divergence ◽

Ex Vivo ◽

Inbred Mouse ◽

Critical Role ◽

Inbred Strains ◽

Mouse Strains ◽

Inbred Mouse Strains ◽

Cardiac Decompensation

Inbred mouse strains play a critical role in biomedical research. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. Here, we compared the cardiac function of C57BL/6J mice to seven other commonly used inbred mouse strains: FVB/NJ, DBA/2J, C3H/HeJ, BALB/cJ, 129X1/SvJ, C57BL/10SnJ, and 129S1/SvImJ. The assays used to compare cardiac function were the ex vivo isolated Langendorff heart preparation and in vivo real-time hemodynamic analysis using conductance micromanometry. We report significant strain-dependent differences in cardiac function between C57BL/6J and other commonly used inbred strains. C57BL/6J maintained better cardiac function than most inbred strains after ex vivo ischemia, particularly compared with 129S1/SvImJ, 129X1/SvJ, and C57BL/10SnJ strains. However, during in vivo acute hypoxia 129X1/SvJ and 129S1/SvImJ maintained relatively normal cardiac function, whereas C57BL/6J animals showed dramatic cardiac decompensation. Additionally, C3H/HeJ showed rapid and marked cardiac decompensation in response to esmolol infusion compared with effects of other strains. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac function. These results may help inform analysis of gene ablation or transgenic studies and further demonstrate specific quantitative traits that could be useful in discovery of genetic modifiers relevant to cardiac health and disease.

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Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0039-1692219 ◽

2019 ◽

Vol 119 (09) ◽

pp. 1394-1402 ◽

Cited By ~ 1

Author(s):

Barbara Thaler ◽

Philipp J. Hohensinner ◽

Johanna Baumgartner ◽

Patrick Haider ◽

Konstantin A. Krychtiuk ◽

...

Keyword(s):

Ex Vivo ◽

Critical Role ◽

Human Monocyte ◽

In Vivo Model ◽

Monocyte Subsets ◽

Plasminogen Activator Inhibitor 1 ◽

Protease Activated Receptors ◽

Messenger Ribonucleic Acid ◽

Pai 1

AbstractMonocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16− monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16− monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16− monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16− monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.

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Investigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging

BioMed Research International ◽

10.1155/2020/2701345 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16 ◽

Cited By ~ 1

Author(s):

Fatéméh Dubois ◽

Magalie Bénard ◽

Bastien Jean-Jacques ◽

Damien Schapman ◽

Hélène Roberge ◽

...

Keyword(s):

Cancer Cells ◽

Stromal Cells ◽

Cell Imaging ◽

Ex Vivo ◽

Critical Role ◽

Treatment Resistance ◽

Stimulated Emission ◽

Neuroendocrine Cells ◽

Tunneling Nanotubes

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC-3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high-resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, ex vivo, in vivo) and the clinical considerations will also be discussed.

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Characterization of the Cellular Reaction to a Collagen-Based Matrix: An In Vivo Histological and Histomorphometrical Analysis

Materials ◽

10.3390/ma13122730 ◽

2020 ◽

Vol 13 (12) ◽

pp. 2730 ◽

Cited By ~ 1

Author(s):

Samuel Ebele Udeabor ◽

Carlos Herrera-Vizcaíno ◽

Robert Sader ◽

C. James Kirkpatrick ◽

Sarah Al-Maawi ◽

...

Keyword(s):

Mononuclear Cells ◽

Ex Vivo ◽

Test Group ◽

Tissue Reaction ◽

Implantation Site ◽

Control Group ◽

The Matrix ◽

Tissue Reactions ◽

Post Implantation

The permeability and inflammatory tissue reaction to Mucomaix® matrix (MM), a non- cross-linked collagen-based matrix was evaluated in both ex vivo and in vivo settings. Liquid platelet rich fibrin (PRF), a blood concentrate system, was used to assess its capacity to absorb human proteins and interact with blood cells ex vivo. In the in vivo aspect, 12 Wister rats had MM implanted subcutaneously, whereas another 12 rats (control) were sham-operated without biomaterial implantation. On days 3, 15 and 30, explantation was completed (four rats per time-point) to evaluate the tissue reactions to the matrix. Data collected were statistically analyzed using analysis of variance (ANOVA) and Tukey multiple comparisons tests (GraphPad Prism 8). The matrix absorbed the liquid PRF in the ex vivo study. Day 3 post-implantation revealed mild tissue inflammatory reaction with presence of mononuclear cells in the implantation site and on the biomaterial surface (mostly CD68-positive macrophages). The control group at this stage had more mononuclear cells than the test group. From day 15, multinucleated giant cells (MNGCs) were seen in the implantation site and the outer third of the matrix with marked increase on day 30 and spread to the matrix core. The presence of these CD68-positive MNGCs was associated with significant matrix vascularization. The matrix degraded significantly over the study period, but its core was still visible as of day 30 post-implantation. The high permeability and fast degradation properties of MM were highlighted.

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Ex Vivo Expansion of Frozen Cord Blood Products without Selection.

Blood ◽

10.1182/blood.v104.11.2844.2844 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2844-2844

Author(s):

Ian K. McNiece ◽

Jenny Harrington ◽

Joshua Kellner ◽

Jennifer Turney ◽

Elizabeth J. Shpall

Keyword(s):

Cord Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

In Vivo Studies ◽

Adherent Cells ◽

Blood Products ◽

Cd34 Cells ◽

Duke University

Abstract Ex vivo expansion of cord blood products (CB) has been proposed as an approach to increase the number of cells available from a single CB unit. We and others have reported the requirement of CD34 selection for optimal expansion of CB products, however, the selection of frozen CB products results in significant losses of CD34+ cells with a median recovery of 43% (range 6 to 203%, N=40) and low purities resulting in decreased expansion. Therefore we explored approaches to expand CB without prior selection and have described the use of co-culture of CB mononuclear cells (MNC) on mesenchymal stem cells (MSC). In the present study we have evaluated the expansion of clinical CB products (provided by Duke University CB Bank CB). MNC were obtained after ficol separation of RBCs and 10% of the CB product was cultured on preformed layers of MSC in T150 flasks containing 50ml of defined media (Sigma Aldrich) plus 100 ng/ml each of rhSCF, rhG-CSF and rhTpo. After 6 days of culture, the non adherent cells were transferred to a Teflon bag and a further 50 ml of media and GFs added to the flask. Again at day 10, non adherent cells were transferred to the Teflon bag and media and growth factors replaced. At day 12 to 13 of incubation the cells were harvested, washed and total nucleated cell (TNC) counts and progenitor assays performed. In three separate experiments we have achieved greater than 20 fold expansion of TNC with a median of 22, and a median expansion of GM-CFC of 37 fold. Morphologic analysis demonstrated the expanded cells contained high levels of mature neutrophils and neutrophil precursors. In vivo studies in NOD/SCID mice also demonstrated that the expanded cells maintained in vivo engraftment potential. Clinical studies are being designed to evaluate the in vivo potential of CB MNC products expanded on MSC.

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Effects of biomechanical forces on signaling in the cortical collecting duct (CCD)

AJP Renal Physiology ◽

10.1152/ajprenal.00634.2013 ◽

2014 ◽

Vol 307 (2) ◽

pp. F195-F204 ◽

Cited By ~ 19

Author(s):

Rolando Carrisoza-Gaytan ◽

Yu Liu ◽

Daniel Flores ◽

Cindy Else ◽

Heon Goo Lee ◽

...

Keyword(s):

Fluid Shear Stress ◽

Ex Vivo ◽

Collecting Duct ◽

Cation Transport ◽

Mapk Signaling ◽

Collagen Type Iv ◽

Cortical Collecting Duct ◽

Type Iv ◽

Biomechanical Forces

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm2 of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

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Functional disruption of α4 integrin mobilizes bone marrow–derived endothelial progenitors and augments ischemic neovascularization

Journal of Experimental Medicine ◽

10.1084/jem.20050459 ◽

2006 ◽

Vol 203 (1) ◽

pp. 153-163 ◽

Cited By ~ 84

Author(s):

Gangjian Qin ◽

Masaaki Ii ◽

Marcy Silver ◽

Andrea Wecker ◽

Evelyn Bord ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Ex Vivo ◽

Critical Role ◽

Cell Surface Receptor ◽

Ischemic Disease ◽

Angiogenic Therapy ◽

Ischemic Tissue ◽

Surface Receptor

The cell surface receptor α4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of α4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of α4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively α4 integrin–expressing cells. In vivo, a single dose of anti–α4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti–α4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti–α4 integrin ex vivo or collected from α4 integrin–deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that α4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of α4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

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MyD88-dependent changes in the pulmonary transcriptome after infection withChlamydia pneumoniae

Physiological Genomics ◽

10.1152/physiolgenomics.00011.2007 ◽

2007 ◽

Vol 30 (2) ◽

pp. 134-145 ◽

Cited By ~ 25

Author(s):

Nuria Rodríguez ◽

Jörg Mages ◽

Harald Dietrich ◽

Nina Wantia ◽

Hermann Wagner ◽

...

Keyword(s):

Chlamydia Pneumoniae ◽

Inflammatory Responses ◽

Ex Vivo ◽

Critical Role ◽

Lipocalin 2 ◽

Immune Effector ◽

A Genome ◽

Genes Encoding ◽

Cellular Replication

Chlamydia pneumoniae, an intracellular bacterium, causes pneumonia in humans and mice. Toll-like receptors and the key adaptor molecule myeloid differentiation factor-88 (MyD88) play a critical role in inducing immunity against this microorganism and are crucial for survival. To explore the influence of MyD88 on induction of immune responses in vivo on a genome-wide level, wildtype (WT) or MyD88−/−mice were infected with C. pneumoniae on anesthesia, and the pulmonary transcriptome was analyzed 3 days later by microarrays. We found that the infection caused pulmonary cellular infiltration in WT but not MyD88−/−mice. Furthermore, it induced the transcription of 360 genes and repressed 18 genes in WT mice. Of these, 221 genes were not or weakly induced in lungs of MyD88−/−mice. This cluster contains primarily genes encoding for chemokines and cytokines like MIP-1α, MIP-2, MIP-1γ, MCP-1, TNF, and KC and other immune effector molecules like immunoresponsive gene-1 and TLR2. Arginase was highly induced after C. pneumoniae infection and was MyD88 dependent. Genes induced by interferons were abundant in a cluster of 102 genes that were only partially MyD88 dependent. Also, lcn2 (lipocalin-2) and timp1 were represented within this cluster. Interestingly, a set of 37 genes including sprr1a was induced more strongly in MyD88−/−mice, and most of them are involved in the regulation of cellular replication. In summary, ex vivo analysis of the pulmonary transcriptome on infection with C. pneumoniae demonstrated a major impact of MyD88 on inflammatory responses but not on interferon-type responses and identified MyD88-independent genes involved in cellular replication.

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