Interacción de la metaloproteinasa de matriz 9 con células de leucemia linfótica crónica: caracterización bioquímica y funcional

158 p.-63 fig.-11 tab.[EN] Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood of CD5+ B lymphocytes and their progressive infiltration of the bone marrowand secondary lymphoid organs. These migration processes are mediated, among other molecules, by matrix metalloproteinases (MMPs). We and others have reported that proMMP (92 kDa) is the major MMP in CLL cells and plays an important role in CLL migration and invasion. Moreover, in contrast with normal B cells, proMMP‐9 is present at the CLL cell surface. Using immunoprecipitation, cell fractionation and confocal microscopy we have demonstrated that α4β1 integrin and 190 kDa CD44v constitute a docking complex for proMMP‐9 in CLL cells. Binding of proMMP‐9 to this complex inhibited cell migration and induced cell survival, thus contributing to CLL progression. Using proMMP‐9 recombinant proteins containing deletions of various domains, we show that the proMMP‐9 hemopexin domain (PEX9) is required for its interaction with CLL cells. Since this interaction may constitute a therapeutic target in CLL, and to help designing specific inhibitors, we have addressed its detailed study. CLL cells bound to the GST‐PEX9 fusion protein generated here, but not to GST, and this binding was primarily mediated by α4β1 integrin. Upon the preparation of truncated GSTPEX9 forms containing the structural blades D1D2 or D3D4, we have demonstrated that CLL cells also efficiently bound to the GST‐D1D2 and GST‐D3D4 proteins and this binding involved two different receptors: GST‐D3D4 primarily bound to α4β1 integrin, while GST‐D1D2 bound to CD44. Furthermore, both regions, D1D2 and D3D4 inhibited CLL cell migration. By preparing overlapping synthetic peptides spanning the entire PEX9 sequence, we identified two specific cell binding sites: The FPGVPLDHDVFQYREKAYFC sequence, located in D1 and contained in peptide P6 and the FDAIAEWIGNQLYFKDGKYW sequence, located in D4 and contained in peptide P3. Both peptides inhibited cell adhesion to proMMP‐9 and GST‐PEX9 as well as transendothelial migration. Moreover, combination of P3 and P6 synergistically increased the inhibitory effect of the individual peptides. Therefore, P3 and P6 could constitute excellent targets to prevent proMMP‐9 contribution to CLL pathogenesis.Vascular endothelial growth factor (VEGF) is also involved in the extravasation and survival of CLL cells. VEGF is a proangiogenic factor also produced by CLL cells and whose elevated levels correlate with bad prognosis. Our results have demonstrated that addition of VEGF to CLL cells significantly reduced proMMP‐9 expression and in a dose‐dependent manner. Inhibition of VEGFR2 blocked this effect, indicating that VEGF signaled through this receptor. Reduction of proMMP‐9 resulted in inhibition of CLL migration through endothelium or Matrigel, confirming the important role of proMMP‐9 in these processes. RT‐PCR analyses indicated that VEGF regulation of proMMP‐9 was at the transcriptional level. Indeed, VEGFVEGFR2 interaction induced phosphorylation of the transcription factor STAT1 and STAT1 silencing restored proMMP‐9 production and CLL migration. As both, VEGF and proMMP‐9, are abundant components of the LLC microenvironment we have also studied the possible regulation of VEGF by proMMP‐9. CLL cell incubation with proMMP‐9 transcriptionally upregulated VEGF levels. In contrast, cell incubation with GST‐PEX9 reduced VEGF expression. These results were confirmed in vasculogénesis assays, where CLL cells preincubated with proMMP‐9 increased tube formation in co‐cultures with HUVEC cells, while preincubation with GST‐PEX9 inhibited vasculogénesis Additionally, immunoprecipitation analyses indicated a possible association between the receptors for proMMP‐9 (α4β1/CD44 ) and VEGF (VEGFR2), that could potentiate intracellular signaling and CLL cell expansion. Altogether, our results provide novel evidences for the pathogenic role of proMMP‐9 and VEGF in CLL and strongly suggest that both proteins could be good therapeutic targets in this malignancy.[ES] La leucemia linfocítica crónica B (LLC) se caracteriza por la acumulación en sangre periférica de linfocitos B CD5+ y su progresiva infiltración de médula ósea y órganos linfoides secundarios. En estos procesos de migración intervienen, entre otras moléculas, las metaloproteinasas de matriz (MMPs). Nuestro grupo, así como otros investigadores, ha demostrado que la proMMP‐9 (92 kDa) es la principal MMP producida por células LLC y juega un papel esencial en la migracion e invasion de células de LLC. Además, y a diferencia con linfocitos B normales, proMMP‐9 está presente en la membrana de células LLC. Por medio de inmunoprecipitaciones, fraccionamiento celulares y microscopía confocal hemos demostrado que la integrina α4β1 y 190 kDa CD44v constituyen un complejo receptor de proMMP‐9 en células LLC. La unión de proMMP‐9 a este complejo inhibía la migración de células LLC e inducía supervivencia celular, contribuyendo por tanto a la progresión de la LLC. Utilizando proMMP‐9 recombinantes con delecciones en varios dominios, hemos demostrado que el dominio hemopexina de proMMP‐9 (PEX9) es necesario para su interacción con células LLC. Dado que que esta interacción puede constituir una buena diana terapéutica en la LLC y con el fin de poder diseñar inhibidores específicos, hemos abordado su estudio en detalle. Las células LLC se unían a la proteína de fusión GST‐PEX9 generada en este estudio, pero no a GST, y esta unión era fundamentalmente mediada por la integrina α4β1. Mediante la preparación de formas truncadas de GST‐PEX9 que contenían los subdominios estructurales D1D2 o D3D4, hemos demostrado que las células LLC se unían también eficazmente a las proteínas GST‐D1D2 y GST‐D3D4 y que esta unión era a través de receptores celulares diferentes: GST‐D3D4 interaccionaba principalmente con la integrina α4β1 mientras que GSTD1D2 se unía a CD44. Además, ambas regiones, D1D2 y D3D4 inhibían la migración de células LLC. Por medio de la preparación de péptidos sintéticos solapantes que abarcaban toda la secuencia de PEX9, hemos identificado dos sitios específicos de unión a células: La secuencia FPGVPLDHDVFQYREKAYFC localizada en el subdominio D4 y contenida en el péptido P3, y la secuencia FDAIAEWIGNQLYFKDGKYW del subdominio D1, contenida en el péptido P6. Ambos péptidos inhibían la adhesión celular a GST‐PEX9 y proMMP‐9 y la migración transendotelial. Además, la combinación de P3 y P6 aumentaba sinergísticamente el efecto inhibidor de cualquiera de ellos. Los sitios P3 y P6 podrían constituir por tanto unas dianas excelentes para prevenir la contribución de proMMP‐9 a la patogénesis de la LLC.Otra molécula implicada en la extravasación y supervivencia de células LLC es el factor de crecimiento endotelial vascular (VEGF). VEGF es un factor proangiogénico que, al igual que proMMP‐9, es producido por células de LLC en niveles elevados, siendo un indicador de mal pronóstico. Nuestros resultados han demostrado que la adición de VEGF a células LLC reducía significativamente, y de manera dosis dependiente, la expresión de proMMP‐9. Este efecto se bloqueaba inhibiendo VEGFR2, indicando que este era el receptor implicado en la señalización por VEGF. La disminución de proMMP‐9 conllevaba una inhibición de la migración transendotelial o a través de Matrigel, confirmando el importante papel de proMMP‐9 en estos procesos. Análisis por RT‐PCR indicaron que la regulación de proMMP‐9 por VEGF era a nivel transcripcional. De hecho, la interacción VEGF‐VEGFR2 inducía la fosforilación del factor de transcripción STAT1 y el silenciamiento de STAT1 restauraba la producción de proMMP‐9 y la migración de células LLC. Dado que tanto VEGF como proMMP‐9 son componentes abundantes del microambiente de la LLC hemos estudiado también la posible regulación de VEGF por proMMP‐9. La incubación de células LLC con proMMP‐9 aumentaba los niveles de VEGF a nivel transcripcional. Por el contrario, la incubación con GST‐PEX9 disminuía la expresión de VEGF. Estos resultados fueron corroborados en ensayos de vasculogénesis, donde células LLC preincubadas con proMMP‐9 aumentaron la formación de tubos en cocultivos con células HUVEC, mientras que la preincubación celular con GST‐PEX9 inhibía la vasculogénesis. Adicionalmente, análisis por inmunoprecipitación indicaron una posible asociación entre los receptores de proMMP‐9 (α4β1/CD44) y VEGF (VEGFR2), que podría conllevar una potenciación de la señalización intracelular y la expansión de células LLC. En conjunto, nuestros resultados aportan nuevas evidencias del papel patogénico de proMMP‐9 y VEGF en la LLC y apoyan que ambas proteínas pueden ser buenas dianas terapéuticas en esta patología.Peer reviewe

Interacción de la metaloproteinasa de matriz 9 con células de leucemia linfocítica crónica: caracterización bioquímica funcional

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 18-06-2014Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood of CD5+ B lymphocytes and their progressive infiltration of the bone marrow and secondary lymphoid organs. These migration processes are mediated, among other molecules, by matrix metalloproteinases (MMPs). We and others have reported that proMMP (92 kDa) is the major MMP in CLL cells and plays an important role in CLL migration and invasion. Moreover, in contrast with normal B cells, proMMP‐9 is present at the CLL cell surface. Using immunoprecipitation, cell fractionation and confocal microscopy we have demonstrated that α4β1 integrin and 190 kDa CD44v constitute a docking complex for proMMP‐9 in CLL cells. Binding of proMMP‐9 to this complex inhibited cell migration and induced cell survival, thus contributing to CLL progression. Using proMMP‐9 recombinant proteins containing deletions of various domains, we show that the proMMP‐9 hemopexin domain (PEX9) is required for its interaction with CLL cells. Since this interaction may constitute a therapeutic target in CLL, and to help designing specific inhibitors, we have addressed its detailed study. CLL cells bound to the GST‐PEX9 fusion protein generated here, but not to GST, and this binding was primarily mediated by α4β1 integrin. Upon the preparation of truncated GSTPEX9 forms containing the structural blades D1D2 or D3D4, we have demonstrated that CLL cells also efficiently bound to the GST‐D1D2 and GST‐D3D4 proteins and this binding involved two different receptors: GST‐D3D4 primarily bound to α4β1 integrin, while GST‐D1D2 bound to CD44. Furthermore, both regions, D1D2 and D3D4 inhibited CLL cell migration. By preparing overlapping synthetic peptides spanning the entire PEX9 sequence, we identified two specific cell binding sites: The FPGVPLDHDVFQYREKAYFC sequence, located in D1 and contained in peptide P6 and the FDAIAEWIGNQLYFKDGKYW sequence, located in D4 and contained in peptide P3. Both peptides inhibited cell adhesion to proMMP‐9 and GST‐PEX9 as well as transendothelial migration. Moreover, combination of P3 and P6 synergistically increased the inhibitory effect of the individual peptides. Therefore, P3 and P6 could constitute excellent targets to prevent proMMP‐9 contribution to CLL pathogenesis. Vascular endothelial growth factor (VEGF) is also involved in the extravasation and survival of CLL cells. VEGF is a proangiogenic factor also produced by CLL cells and whose elevated levels correlate with bad prognosis. Our results have demonstrated that addition of VEGF to CLL cells significantly reduced proMMP‐9 expression and in a dose‐dependent manner. Inhibition of VEGFR2 blocked this effect, indicating that VEGF signaled through this receptor. Reduction of proMMP‐9 resulted in inhibition of CLL migration through endothelium or Matrigel, confirming the important role of proMMP‐9 in these processes. RT‐PCR analyses indicated that VEGF regulation of proMMP‐9 was at the transcriptional level. Indeed, VEGFVEGFR2 interaction induced phosphorylation of the transcription factor STAT1 and STAT1 silencing restored proMMP‐9 production and CLL migration. As both, VEGF and proMMP‐9, are abundant components of the LLC microenvironment we have also studied the possible regulation of VEGF by proMMP‐9. CLL cell incubation with proMMP‐9 transcriptionally upregulated VEGF levels. In contrast, cell incubation with GST‐PEX9 reduced VEGF expression. These results were confirmed in vasculogénesis assays, where CLL cells preincubated with proMMP‐9 increased tube formation in co‐cultures with HUVEC cells, while preincubation with GST‐PEX9 inhibited vasculogénesis Additionally, immunoprecipitation analyses indicated a possible association between the receptors for proMMP‐9 (α4β1/CD44 ) and VEGF (VEGFR2), that could potentiate intracellular signaling and CLL cell expansion. Altogether, our results provide novel evidences for the pathogenic role of proMMP‐9 and VEGF in CLL and strongly suggest that both proteins could be good therapeutic targets in this malignancy.La leucemia linfocítica crónica B (LLC) se caracteriza por la acumulación en sangre periférica de linfocitos B CD5+ y su progresiva infiltración de médula ósea y órganos linfoides secundarios. En estos procesos de migración intervienen, entre otras moléculas, las metaloproteinasas de matriz (MMPs). Nuestro grupo, así como otros investigadores, ha demostrado que la proMMP‐9 (92 kDa) es la principal MMP producida por células LLC y juega un papel esencial en la migracion e invasion de células de LLC. Además, y a diferencia con linfocitos B normales, proMMP‐9 está presente en la membrana de células LLC. Por medio de inmunoprecipitaciones, fraccionamiento celulares y microscopía confocal hemos demostrado que la integrina α4β1 y 190 kDa CD44v constituyen un complejo receptor de proMMP‐9 en células LLC. La unión de proMMP‐9 a este complejo inhibía la migración de células LLC e inducía supervivencia celular, contribuyendo por tanto a la progresión de la LLC. Utilizando proMMP‐9 recombinantes con delecciones en varios dominios, hemos demostrado que el dominio hemopexina de proMMP‐9 (PEX9) es necesario para su interacción con células LLC. Dado que que esta interacción puede constituir una buena diana terapéutica en la LLC y con el fin de poder diseñar inhibidores específicos, hemos abordado su estudio en detalle. Las células LLC se unían a la proteína de fusión GST‐PEX9 generada en este estudio, pero no a GST, y esta unión era fundamentalmente mediada por la integrina α4β1. Mediante la preparación de formas truncadas de GST‐PEX9 que contenían los subdominios estructurales D1D2 o D3D4, hemos demostrado que las células LLC se unían también eficazmente a las proteínas GST‐D1D2 y GST‐D3D4 y que esta unión era a través de receptores celulares diferentes: GST‐D3D4 interaccionaba principalmente con la integrina α4β1 mientras que GSTD1D2 se unía a CD44. Además, ambas regiones, D1D2 y D3D4 inhibían la migración de células LLC. Por medio de la preparación de péptidos sintéticos solapantes que abarcaban toda la secuencia de PEX9, hemos identificado dos sitios específicos de unión a células: La secuencia FPGVPLDHDVFQYREKAYFC localizada en el subdominio D4 y contenida en el péptido P3, y la secuencia FDAIAEWIGNQLYFKDGKYW del subdominio D1, contenida en el péptido P6. Ambos péptidos inhibían la adhesión celular a GST‐PEX9 y proMMP‐9 y la migración transendotelial. Además, la combinación de P3 y P6 aumentaba sinergísticamente el efecto inhibidor de cualquiera de ellos. Los sitios P3 y P6 podrían constituir por tanto unas dianas excelentes para prevenir la contribución de proMMP‐9 a la patogénesis de la LLC. Otra molécula implicada en la extravasación y supervivencia de células LLC es el factor de crecimiento endotelial vascular (VEGF). VEGF es un factor proangiogénico que, al igual que proMMP‐9, es producido por células de LLC en niveles elevados, siendo un indicador de mal pronóstico. Nuestros resultados han demostrado que la adición de VEGF a células LLC reducía significativamente, y de manera dosis dependiente, la expresión de proMMP‐9. Este efecto se bloqueaba inhibiendo VEGFR2, indicando que este era el receptor implicado en la señalización por VEGF. La disminución de proMMP‐9 conllevaba una inhibición de la migración transendotelial o a través de Matrigel, confirmando el importante papel de proMMP‐9 en estos procesos. Análisis por RT‐PCR indicaron que la regulación de proMMP‐9 por VEGF era a nivel transcripcional. De hecho, la interacción VEGF‐VEGFR2 inducía la fosforilación del factor de transcripción STAT1 y el silenciamiento de STAT1 restauraba la producción de proMMP‐9 y la migración de células LLC. Dado que tanto VEGF como proMMP‐9 son componentes abundantes del microambiente de la LLC hemos estudiado también la posible regulación de VEGF por proMMP‐9. La incubación de células LLC con proMMP‐9 aumentaba los niveles de VEGF a nivel transcripcional. Por el contrario, la incubación con GST‐PEX9 disminuía la expresión de VEGF. Estos resultados fueron corroborados en ensayos de vasculogénesis, donde células LLC preincubadas con proMMP‐9 aumentaron la formación de tubos en cocultivos con células HUVEC, mientras que la preincubación celular con GST‐PEX9 inhibía la vasculogénesis. Adicionalmente, análisis por inmunoprecipitación indicaron una posible asociación entre los receptores de proMMP‐9 (α4β1/CD44) y VEGF (VEGFR2), que podría conllevar una potenciación de la señalización intracelular y la expansión de células LLC. En conjunto, nuestros resultados aportan nuevas evidencias del papel patogénico de proMMP‐9 y VEGF en la LLC y apoyan que ambas proteínas pueden ser buenas dianas terapéuticas en esta patologí

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

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

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

The biological potential hidden in inclusion bodies

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes

Recombinant protein-based nanoparticles : elucidating their inflammatory effects in vivo and their potential as a new therapeutic format

Altres ajuts: we are also indebted to CERCA Programme (Generalitat de Catalunya) and European Social Fund for supporting our research. L.G.-R. received a pre-doctoral fellowship from INIA (FPI-INIA, MINECO), R.R.-P. from AGAUR (FI-AGAUR), and O.C.-G. from MECD (FPU). E.G.-F. received a post-doctoral fellowship from INIA (DOC-INIA) and A.V. an ICREA Academia award.Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties-intrinsic to IBs-with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format

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

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

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.status: publishe

VEGF/VEGFR2 interaction down-regulates matrix metalloproteinase–9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration

Brief Report. Artículo breve.B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules, including matrix metalloproteinase–9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Down-regulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or human umbilical vein endothelial cells, confirming the crucial role of MMP-9 in these processes. Reverse-transcription polymerase chain reaction analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation, and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 down-regulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.Ministerio de Ciencia e Innovación, Spain (grants PI060400, SAF2009-07035, and RTICC RD06/0020/0011, A.G.-P.; and PI061637 and RTICC RD06/0020/0080, M.J.T.) and by the Fundación de Investigación Médica Mutua Madrileña (A.G.-P.). J.R.-M. was supported by the Fundación Ramón Areces. P.E. was supported by the Ministerio de Ciencia e Innovación.Peer reviewe