Inhibition of MMP-9-dependent degradation of gelatin, but not other MMP-9 substrates, by the MMP-9 hemopexin domain blades 1 and 4

11 p.-5 fig.-1 tab.Degradation and remodeling of the extracellular matrix by matrix metalloproteinases (MMPs) plays important roles in normal development, inflammation, and cancer. MMP-9 efficiently degrades the extracellular matrix component gelatin, and the hemopexin domain of MMP-9 (PEX9) inhibits this degradation. To study the molecular basis of this inhibition, we generated GST fusion proteins containing PEX9 or truncated forms corresponding to specific structural blades (B1-B4) of PEX9. GST-PEX9 inhibited MMP-9-driven gelatin proteolysis, measured by gelatin zymography, FITC-gelatin conversion, and DQ-gelatin degradation assays. However, GST-PEX9 did not prevent the degradation of other MMP-9 substrates, such as a fluorogenic peptide, αB crystalline, or nonmuscular actin. Therefore, PEX9 may inhibit gelatin degradation by shielding gelatin and specifically preventing its binding to MMP-9. Accordingly, GST-PEX9 also abolished the degradation of gelatin by MMP-2, confirming that PEX9 is not an MMP-9 antagonist. Moreover, GST-B4 and, to a lesser extent, GST-B1 also inhibited gelatin degradation by MMP-9, indicating that these regions are responsible for the inhibitory activity of PEX9. Accordingly, ELISAs demonstrated that GST-B4 and GST-B1 specifically bound to gelatin. Our results establish new functions of PEX9 attributed to blades B4 and B1 and should help in designing specific inhibitors of gelatin degradation.This work was supported by Grant SAF2012-31613 and Red Temática de Investigación Cooperativa en Cáncer Grant RD12/0036/0061 from the Ministry of Economy and Competitivity (Spain) (to A. G.-P.); by Grant S2010/ BMD-2314 (to A. G.-P.) from the Comunidad de Madrid/European Union;and by the Concerted Research Actions Grant GOA 2013–2015 and the fund for Scientific Research of Flanders (to G. O.).Peer reviewe

Effectiveness of a dog-assisted therapy programme to enhance quality of life in institutionalized dementia patients

The aim of this study was to assess the effectiveness of a dog-assisted therapy intervention to enhance quality of life in persons with dementia institutionalized in a public care home and to study its effect on the use of psychotropic medications. A dog-assisted therapy intervention was designed, in which 34 residents of a public care home in Cuenca (Spain) participated. The participants were assigned to two groups, an experimental and a control group. The program consisted of one 50-min session per week during 9months. Analysis of covariance (ANCOVA) was used to compare post-training values between groups, using baseline values as covariates. Our findings do seem to support the hypothesis that Animal-Assisted Therapy may contribute to enhancing quality of life for residents in an aged care home. However, aspects such as the format, time, and content of the sessions as well as their possibilities of reducing psychotropic medication require more research

Molecular Players in Hematologic Tumor Cell Trafficking

The trafficking of neoplastic cells represents a key process that contributes to progression of hematologic malignancies. Diapedesis of neoplastic cells across endothelium and perivascular cells is facilitated by adhesion molecules and chemokines, which act in concert to tightly regulate directional motility. Intravital microscopy provides spatio-temporal views of neoplastic cell trafficking, and is crucial for testing and developing therapies against hematologic cancers. Multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL) are hematologic malignancies characterized by continuous neoplastic cell trafficking during disease progression. A common feature of these neoplasias is the homing and infiltration of blood cancer cells into the bone marrow (BM), which favors growth and survival of the malignant cells. MM cells traffic between different BM niches and egress from BM at late disease stages. Besides the BM, CLL cells commonly home to lymph nodes (LNs) and spleen. Likewise, ALL cells also infiltrate extramedullary organs, such as the central nervous system, spleen, liver, and testicles. The α4β1 integrin and the chemokine receptor CXCR4 are key molecules for MM, ALL, and CLL cell trafficking into and out of the BM. In addition, the chemokine receptor CCR7 controls CLL cell homing to LNs, and CXCR4, CCR7, and CXCR3 contribute to ALL cell migration across endothelia and the blood brain barrier. Some of these receptors are used as diagnostic markers for relapse and survival in ALL patients, and their level of expression allows clinicians to choose the appropriate treatments. In CLL, elevated α4β1 expression is an established adverse prognostic marker, reinforcing its role in the disease expansion. Combining current chemotherapies with inhibitors of malignant cell trafficking could represent a useful therapy against these neoplasias. Moreover, immunotherapy using humanized antibodies, CAR-T cells, or immune check-point inhibitors together with agents targeting the migration of tumor cells could also restrict their survival. In this review, we provide a view of the molecular players that regulate the trafficking of neoplastic cells during development and progression of MM, CLL, and ALL, together with current therapies that target the malignant cells

Sphingosine-1-phosphate activates chemokine-promoted myeloma cell adhesion and migration involving α4β1 integrin function

51 p.-7 fig.-1 tab.-2 fig.supl.Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1-dependent myeloma cell adhesion is up-regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine-1-phosphate (S1P) regulates immune cell trafficking upon binding to G-protein-coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up-regulated the α4β1-mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high-affinity α4β1 that efficiently bound the α4β1 ligand VCAM-1, a finding that was associated with S1P-triggered increase in talin-β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1-dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2-Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1-dependent adhesion and migration of myeloma cells by CXCL12-S1P combined activities might have important consequences for myeloma disease progressionThis study was supported by the Ministerio de Ciencia e Innovación (Grant Nos SAF2011-24022 to JT, SAF2009-07035 to AGP, SAF2009-11037 to AH, RD06/0020/0011 to JT and AGP, RD06/0020/0006 to NCG and MG and PI081825 to MG), the Comunidad de Madrid (Grant No. P2010/BMD-2314 to AGP, JT and AH) and the Fundación de Investigación Médica Mutua Madrileña (to AGP and MG). AH was also funded by the FP7-People-2009-RG (Grant No. 246655), a Ramón y Cajal Fellowship (Grant No. RYC-2007-00697) and the Pro-CNIC Foundation.Peer reviewe

A 17-residue sequence from the matrix metalloproteinase-9 (MMP-9) hemopexin domain binds α4β1 integrin and inhibits MMP-9-induced functions in chronic lymphocytic leukemia B cells

13 páginas, 7 figuras, 2 tablas -- PAGS nros. 27601-27613We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC50 values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesisThis work was supported by Grants SAF2009–07035 and RTICC RD06/0020/0011 (to A. G.-P.) and RTICC RD06/0020/0080 (to M. J. T.) from the Ministerio de Ciencia e Innovación, Spain, and by a grant from the Fundación Puerta de Hierro (to J. A. G. M.)Peer reviewe

AT514, a cyclic depsipeptide from Serratia marcescens, induces apoptosis of B-chronic lymphocytic leukemia cells. Interference with the Akt/NF-kB survival pathway

8 páginas, 5 figuras -- PAGS nros. 572-579Clinical treatment of B-cell chronic lymphocytic leukemia (B-CLL) is limited by the progressive drug resistance and nonselectivity of most drugs towards malignant cells. Depsipeptides are present in certain bacteria and display potent antitumor activity. We have studied the effect of the novel cyclodepsipeptide AT514 (serratamolide) from Serratia marcescens on B-CLL cell viability. AT514 induced apoptosis of B-CLL cells from the 21 patients studied, as confirmed by Annexin-V binding and nuclei condensation, with an average IC50 of 13 M. AT514 was effective in those B-CLL cases resistant to fludarabine, but had no effect on normal PBL. AT514 preferentially activated the intrinsic apoptotic pathway, as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9 and -3, but not of caspase-8. Importantly, AT514 interfered with phosphatidylinositol-3 kinase and protein kinase C survival signals since it increased the apoptotic effect of LY294002 and BisI inhibitors, and induced Akt dephosphorylation at Ser 473. AT514 also decreased NF-B activity by dramatically reducing the levels of p65 in B-CLL. This was confirmed on functional assays using NF-B-luc-transfected Raji cells and transgenic mice. Our results establish that AT514 induces apoptosis of primary B-CLL cells and could be useful for clinical treatment of this malignancyThis work was supported by grants 08.3/0030.1/2003 from the Comunidad Autónoma de Madrid, SAF2003-00824 from the Ministerio de Ciencia y Tecnología (MCyT), and 01/1183 from Fondo de Investigación Sanitaria (to AGP); and CIDEM Grant 301888 (Generalitat de Catalunya)/Fundació Bosch i Gimpera, to RPT). E Escobar and E López-Martín were supported by fellowships from MCyTPeer reviewe

Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction via extracellular signal-regulated kinase-1/2 signaling and is involved in CCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migration

4 páginas, 2 figuras -- PAGS nros. 383-386B-cell chronic lymphocytic leukemia (B-CLL) progression is frequently accompanied by clinical lymphadenopathy, and the CCL21 chemokine may play an important role in this process. Indeed, CCR7 (the CCL21 receptor), as well as matrix metalloproteinase-9 (MMP-9), are overexpressed in infiltrating B-CLL cells. We have studied whether MMP-9 is regulated by CCL21 and participates in CCL21-dependent migration. CCL21 significantly increased B-CLL MMP-9 production, measured by gelatin zymography. This was inhibited by blocking extracellular signal-regulated kinase-1/2 (ERK1/2) activity or by cell transfection with CCR7-siRNA. Accordingly, CCL21/CCR7 interaction activated the ERK1/2/c-Fos pathway and increased MMP-9 mRNA. CCL21-driven B-CLL cell migration through Matrigel or human umbilical vein endothelial cells (HUVEC) was blocked by anti-CCR7 antibodies, CCR7-siRNA transfection, or the ERK1/2 inhibitor U0126, as well as by anti-MMP-9 antibodies or tissue inhibitor of metalloproteinase 1 (TIMP-1). These results strongly suggest that MMP-9 is involved in B-CLL nodal infiltration and expand the roles of MMP-9 and CCR7 in B-CLL progression. Both molecules could thus constitute therapeutic targets for this diseaseThis work was supported by grants PI060400 (to A.G.P.) and PI061637 (to M.J.T.) from the Ministerio de Sanidad y Consumo, and by the Fundación de Investigación Médica Mutua Madrileña (FMM; to A.G.P.). J.R.M. was supported by FMM and the Fundación Ramón ArecesPeer reviewe

Therapeutic effects of psyllium in type 2 diabetic patients

P. 830-842The study included three phases: phase 1 (1 week), phase 2 (treatment, 14 g fibre=day, 6 weeks) and phase 3 (4 weeks). At the end of each phase a clinical evaluation was performed after the ingestion of a test breakfast of 1824.2 kJ (436 kcal). Measurements included concentrations of blood glucose, insulin, fructosamine, GHbA1c, C-peptide and 24 h urinary glucose excretion. In addition, uric acid, cholesterol and several mineral and vitamin concentrations were also evaluated. Twenty type 2 diabetic patients (12 men and 8 women) participated in the study with a mean age of 67.4 y for men and 66 y for women. The mean body mass index of men was 28.2 kg=m2 and that of women 25.9 kg=m2. Glucose absorption decreased significantly in the presence of psyllium (12.2%); this reduction is not associated with an important change in insulin levels (5%). GHbA1c, C-peptide and 24 h urinary glucose excretion decreased (3.8, 14.9 and 22.5%, respectively) during the treatment with fibre (no significant differences) as well as fructosamine (10.9%, significant differences). Psyllium also reduced total and LDL cholesterol (7.7 and 9.2%, respectively, significant differences), and uric acid (10%, significant difference). Minerals and vitamins did not show important changes, except sodium that increased significantly after psyllium administration.S

Matrix metalloproteinase-9 induces a pro-angiogenic profile in chronic lymphocytic leukemia cells

27 p.-3 fig.-2 tab.Increased angiogenesis is commonly observed in chronic lymphocytic leukemia (CLL) tissues in correlation with advanced disease. CLL cells express pro- and anti-angiogenic genes and acquire a pro-angiogenic pattern upon interaction with the microenvironment. Because MMP-9 (a microenvironment component) plays important roles in solid tumor angiogenesis, we have studied whether MMP-9 influenced the angiogenic pattern in CLL cells. Immunofluorescence analyses confirmed the presence of MMP-9 in CLL tissues. MMP-9 interaction with CLL cells increased their MMP-9 expression and secretion into the medium. Accordingly, the conditioned media of MMP-9-primed CLL cells significantly enhanced endothelial cell proliferation, compared to control cells. MMP-9 also increased VEGF and decreased TSP-1 and Ang-2 expression, all at the gene and protein level, inducing a pro-angiogenic pattern in CLL cells. Mechanistic analyses demonstrated that downregulation of the selected gene TSP-1 by MMP-9 involved α4β1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology.This work was supported by grants SAF2015-69180R from the Ministerio de Economía y Competitividad (Spain)/Fondo Europeo de Desarrollo Regional (FEDER), European Union; S2010/BMD-2314 from the Comunidad de Madrid/European Union (AGP.); and by the Concerted Research Actions (KU Leuven C1 Grant C16/17/010) and the Research Foundation of Flanders (FWO-Vlaanderen, to EUB, PEVdS, and GO).Peer reviewe