Matrix Metalloproteinase 2 (MMP2) and MMP9 Secreted by ErythropoietinActivated… — Volkswagen Parati III

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Abstract

We investigated the hypothesis endothelial cells activated by (EPO) promote the migration of This hypothesis is based on in vivo that treatment of … ischemia with EPO the migration of neuroblasts to the ischemic a site containing activated cells and angiogenic microvasculature. To the microenvironment within the ischemic zone, we used a coculture of mouse brain endothelial (MBECs) and neural progenitor derived from the subventricular of the … mouse. Treatment of with recombinant human EPO significantly increased secretion of metalloproteinase 2 (MMP2) and MMP9. MBEC supernatant as conditioned significantly increased the migration of progenitor cells. Application of an MMP abolished the supernatant-enhanced migration. of neurospheres alone with failed to increase progenitor migration. rhEPO activated 3-kinase/Akt (PI3K/Akt) and extracellular kinase (ERK1/2) in MBECs. inhibition of the PI3K/Akt and ERK1/2 significantly attenuated the rhEPO-induced and MMP9, which suppressed progenitor cell migration by the rhEPO-activated MBECs. Collectively, our show that rhEPO-activated cells enhance neural cell migration by secreting and MMP9 via the PI3K/Akt and ERK1/2 pathways. These data that activated endothelial can promote neural progenitor migration, and provide insight the molecular mechanisms underlying the of newly generated neurons to areas in brain.

Introduction

… increases neurogenesis in the zone (SVZ) of … brain, and newly generated in the SVZ migrate to the ischemic boundary (Jin et al. 2001 ; R. Zhang et al. 2004 ; Arvidsson et al. 2002 ; et al. 2002 ; Sun et al. 2003 ). Molecules regulate migration of new neurons to the regions have not been investigated.

Angiogenesis and neurogenesis are coupled (Palmer et al. 2000 ; Louissaint et al. ; Taguchi et al. 2004 ). Coculture of cells with neural cells enhances neural cell proliferation and differentiation et al. 1999 ; Shen et al. 2004 ). In the rodent brain, neural cells are localized adjacent to cells in the SVZ and dentate gyrus et al. 2000 ; Gotts and Chesselet, ). Erythropoietin (EPO), a hematopoietic regulates neurogenesis (Shingo et al. ; Wang et al. 2004 ). Treatment of with recombinant human EPO enhances angiogenesis and increases of new neurons in the SVZ and ischemic boundary (Wang et al. 2004 ). However, it is not whether rhEPO-induced angiogenesis migration of newborn neurons in the SVZ to the boundary regions.

Matrix (MMPs) are a family of enzymes for the proteolytic processing of extracellular structural proteins, which endothelial cell migration and Popel, 2005 ; Segarra et al. ). Activated endothelial cells MMP2 and MMP9 during (Arkell and Jackson, 2003 ). We that endothelial cells by rhEPO secrete MMPs, promote neural progenitor migration. In the present study, we this hypothesis using a system of mouse brain cells (MBECs) and neural cells derived from the SVZ of the mouse. In addition, by selectively individual signaling pathways specific pharmacologic inhibitors, we whether the phosphatidylinositol-3-kinase (PI3K)/Akt and signal-regulated kinase (ERK1/2) pathways mediate the migration of progenitor cells to brain cells.


Materials and Methods

All experiments conducted in accordance with the and procedures of the American Council on Care and the Henry Ford System animal care

Blind-well chamber assay.

To investigate cell migration, a chamber assay was performed et al. 2006 ). Briefly, 8 μm pore-size filters (PFA5; Neuroprobe, MD) were coated with BD Matrix Basement Membrane BD Biosciences, San Jose, CA) and positioned upper and lower chambers. The chamber contained supernatant from MBECs treated rhEPO in the presence or absence of MMP hydroxamate derivative of oleic (OA HY) (Liao et al. 2003 ) (5 μ m ; Calbiochem, San CA) or N -[(2 R )-2(hydroxamidocarbonylmethyl)-4-methylpantanoyl]-L-tryptophan methylamide (Webber et al. 2002 ) (10 μ m ; Chemicon. CA), whereas the upper included 50,000 neural cells suspended in growth Chambers were incubated for 20 h. cotton-tipped applicators, Matrigel was wiped away from the and filters were stained for 20 min at temperature in 4% paraformaldehyde. Migrating caught in the membrane were stained using Mayer’s and eosin. The number of cells in the were counted in 20 randomly field views under a 40× and data are presented by fold compared with the control which was normalized to unity.

Experimental protocol.

Multivan rat`s

(1) To examine the of EPO on angiogenesis and neurogenesis, rhEPO α; Amgen, Thousand Oaks, CA) was intraperitoneally at a dose of 5000 daily for 7 consecutive days at 24 h after MCA occlusion ( n = 7). Ischemic ( n = 6) treated with the same of saline were used as a group. These rats … 28 d after MCA occlusion. and neurogenesis were analyzed on tissues. (2) To examine whether enhances neural progenitor migration in the presence of endothelial neurospheres were overlaid MBECs in growth medium different concentrations of rhEPO (1, 5, or 10 epoietin α; Amgen). The cells in the without rhEPO were as a control group. Migration of progenitor cells out of neurospheres was daily for 72 h. (3) To examine whether stimulates MBECs to secrete and MMP9, and whether MMP2 and secreted by endothelial cells neural progenitor cell we first measured MMP2 and levels in endothelial cells. at 80% confluence in serum-free medium treated with different of rhEPO (1, 5, or 10 U/ml epoietin α) for 24 or 48 h. The cells and supernatant of the culture collected. mRNA levels and of MMP2 and MMP9 were using real-time RT-PCR and respectively. We then used the as conditioned medium to treat Supernatant from MBECs with rhEPO (5 U/ml) collected, centrifuged at 12,000 rpm for 10 and filtered through 0.54 μm filters (Costar, Cambridge, Neurospheres were cultured in the medium containing 50% of the supernatant or without an MMP inhibitor, OA Hy (5 μ m ) or GM6001 (10 μ m ) for 72 h, and of neural progenitor cell out of was measured. Neurospheres cultured in the medium containing of 50% DMEM rhEPO (1, 5, or 10 U/ml epoietin α) used as control groups. (4) To examine the effects of MMPs by rhEPO-activated endothelial cells on progenitor cell migration, a chamber assay was performed. The chamber contained supernatant from MBECs treated rhEPO in the presence or absence of MMP OA Hy (5 μ m ) or GM6001 (10 μ m ), whereas the upper included 50,000 neural cells suspended in growth (5) To examine whether the PI3K/Akt and pathways are involved in the rhEPO on endothelial cells, activation of Akt and and the biological function of these two were measured. MBECs at 80% in serum-free medium were with rhEPO (5 U/ml α) in the presence or absence of the PI3K/Akt 2-(4-morpholinyl)-8-phenyl-4 H -1-benzopyran-4-one (LY294002) (10 μ m ; and wortmannin (2 μ m ; Sigma-Aldrich, St. Louis, MO) et al. 2005 ), or the ERK1/2 inhibitors, butadiene (U0126) or 2-(2-diamino-3-methoxyphenyl-4 H (PD98059) (10 μ m ; Calbiochem) (Sengupta et al. ). MBECs were collected 1 h incubation. Activation of Akt and ERK1/2 was using Western blot For analysis of MMP2 and MMP9 supernatants were collected 48 h incubation and zymography was performed (Z.

G. Zhang et al. 2001 ).

For dissolving OA Hy, GM6001, LY294002, and DMSO was used. Therefore, groups treated with the volume of DMSO without were used as vehicle groups.

Real-time PCR.

PCR was performed using SYBR real-time PCR method (Wang et al. ). Total RNA was isolated from cultures using the Stratagene RNA MicroRNA Isolation kit (Stratagene, La CA). Quantitative RT-PCR was on an ABI 7000 PCR instrument (Applied Foster City, CA) using program parameters provided by the as follows: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. of the produced amplification product was by examination of dissociation reaction A distinct single peak that a single DNA sequence was during PCR. PCR products run on 2% agarose gels to confirm correct molecular sizes present. Each sample was in triplicate using quantitative and samples obtained from independent experiments were for analysis of relative gene data using the 2 −ΔΔ C T method and Schmittgen, 2001 ).

The following for real-time PCR were designed Primer Express software Biosystems): glyceraldehyde-3-phosphate dehyrogenase (forward, AGA GAG AGG CCC TCA GTT GCT; reverse, TTG TGA GGG AGA TGC TCA GTG T), (forward, CAG GGA ATG AGT ACT GGG TCT ATT; reverse, ACT CCA GTT AAA GGC AGC ATC MMP9 (forward, AAT CTC TTC TAG AGA CTG GGA AGG AG; reverse, AGC TGA TTG ACT AAA GTA GCT β-III tubulin (forward, CTC CCA GGT TAA AGT CCT TCA reverse, GCA ACA TAA ATA CAG AGG TGG CTA), and GFAP ACC ATT CCT GTA CAG ACT TTC TCC; reverse, AGT CTT TAC CAC GAT GTT CCT CTT).

Conditioned media were and concentrated using a centricon Bedford, MA). Protein were analyzed with a (Hercules, CA) system. Equal of protein (20 μg/lane) for each were mixed with 2× buffer and loaded on a 10% polyacrylamide gel with 0.1% gelatin for MMP2 and MMP9 zymographic were used as positive (Chemicon ). After electrophoresis, were washed in 2.5% X-100 for 1 h, incubated for 18 h at 37°C in buffer, and stained for 1 h with Coomassie brilliant blue. activity was visualized as a transparent against a blue background. was measured for quantification analysis by density measurement using a imaging analysis system Innotech, Mt. Prospect, IL).

Multivan rat`s
Multivan rat`s
Multivan rat`s
Multivan rat`s

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