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1.
Int J Toxicol ; 40(4): 344-354, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33866838

RESUMO

Phosphatidylinositol 3-kinase (PI3K) δ is a lipid kinase primarily found in leukocytes, which regulates important cell functions. AMG2519493 was a PI3K δ-specific inhibitor in development for treatment of various inflammatory diseases. AMG2519493-related changes in the male and/or female reproductive organs were observed in the 1- and 3-month oral repeat dose toxicology studies in the rat and cynomolgus monkey. Hemorrhagic corpora lutea cysts and increased incidence of corpora lutea cysts without hemorrhage were observed in the ovaries at supra pharmacological doses in the rat. A decrease in seminiferous germ cells in the testis, indicative of spermatogenesis maturation arrest, was observed in both the rat and cynomolgus monkey. Although the characteristics were comparable, the drug systemic exposures associated with the testicular changes were very different between the 2 species. In the rat, the testicular change was only observed at supra pharmacologic exposure. Isotype assessment of PI3K signaling in rat spermatogonia in vitro indicated a role for PI3K ß, but not δ, in the c Kit/PI3K/protein kinase B signaling pathway. Therefore, changes in both the ovary and testis of the rat were considered due to off target effect as they only occurred at suprapharmacologic exposure. In contrast, the testicular changes in the cynomolgus monkey (decrease in seminiferous germ cells) occurred at very low doses associated with PI3K δ-specific inhibition, indicating that the PI3K δ isoform may be important in spermatogenesis maturation in the cynomolgus monkey. Our results suggest species-related differences in PI3K isoform-specific control on reproductive organs.


Assuntos
Ovário/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Testículo/efeitos dos fármacos , Animais , Feminino , Macaca fascicularis , Masculino , Camundongos , Ovário/enzimologia , Ratos , Ratos Sprague-Dawley , Espermatogônias/enzimologia , Testículo/enzimologia
2.
J Immunotoxicol ; 17(1): 110-121, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32525431

RESUMO

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (ß-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and ß-arrestin endpoints and a specificity of 93% (ß-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.


Assuntos
Degranulação Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/efeitos adversos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Antígenos CD34/metabolismo , Degranulação Celular/imunologia , Engenharia Celular , Células Cultivadas , Testes Imunológicos de Citotoxicidade/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Histamina/análise , Histamina/metabolismo , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Cultura Primária de Células , Sensibilidade e Especificidade
3.
Cancer Cell Int ; 14(1): 99, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25317077

RESUMO

BACKGROUND: Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding ß1 integrins in collagen-stimulated MMP-2 activation. METHODS: Three ß1 integrin siRNAs were used to elucidate the involvement of ß1 integrins in the Col I-induced MMP-2 activation mechanism. ß1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of ß1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography. RESULTS: All three ß1 integrin siRNAs were efficient at ß1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of ß1, but not αV, which is not. All three ß1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of ß1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped ß1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the ß1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by ß1 integrin siRNA knockdown. CONCLUSION: Together, the data reveals that strong abrogation of ß1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of ß1 integrin.

4.
Toxicol Pathol ; 41(2): 235-62, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23334697

RESUMO

The proper folding, assembly, and maintenance of cellular proteins is a highly regulated process and is critical for cellular homeostasis. Multiple cellular compartments have adapted their own systems to ensure proper protein folding, and quality control mechanisms are in place to manage stress due to the accumulation of unfolded proteins. When the accumulation of unfolded proteins exceeds the capacity to restore homeostasis, these systems can result in a cell death response. Unfolded protein accumulation in the endoplasmic reticulum (ER) leads to ER stress with activation of the unfolded protein response (UPR) governed by the activating transcription factor 6 (ATF6), inositol requiring enzyme-1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK) signaling pathways. Many xenobiotics have been shown to influence ER stress and UPR signaling with either pro-survival or pro-death features. The ultimate outcome is dependent on many factors including the mechanism of action of the xenobiotic, concentration of xenobiotic, duration of exposure (acute vs. chronic), cell type affected, nutrient levels, oxidative stress, state of differentiation, and others. Assessing perturbations in activation or inhibition of ER stress and UPR signaling pathways are likely to be informative parameters to measure when analyzing mechanisms of action of xenobiotic-induced toxicity.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Xenobióticos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dobramento de Proteína , Transdução de Sinais , Resposta a Proteínas não Dobradas/efeitos dos fármacos
5.
J Cell Sci ; 123(Pt 21): 3808-16, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20940254

RESUMO

Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.


Assuntos
Transdiferenciação Celular , Endotélio Vascular/metabolismo , Vasos Linfáticos/metabolismo , Células Progenitoras Linfoides/metabolismo , Microtúbulos/metabolismo , Adulto , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Colágeno/metabolismo , Combinação de Medicamentos , Endotélio Vascular/patologia , Matriz Extracelular , Fibrina/metabolismo , Humanos , Laminina/metabolismo , Vasos Linfáticos/patologia , Células Progenitoras Linfoides/patologia , Neovascularização Fisiológica , Fenótipo , Proteoglicanas/metabolismo , Engenharia Tecidual
6.
Mol Biol Cell ; 20(7): 2030-40, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19211836

RESUMO

Membrane type-1 matrix metalloproteinase (MT1-MMP) supports tumor cell invasion through extracellular matrix barriers containing fibrin, collagen, fibronectin, and other proteins. Here, we show that simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, and TSPAN12) markedly decreases MT1-MMP proteolytic functions in cancer cells. Affected functions include fibronectin proteolysis, invasion and growth in three-dimensional fibrin and collagen gels, and MMP-2 activation. Tetraspanin proteins (CD9, CD81, and TSPAN2) selectively coimmunoprecipitate and colocalize with MT1-MMP. Although tetraspanins do not affect the initial biosynthesis of MT1-MMP, they do protect the newly synthesized protein from lysosomal degradation and support its delivery to the cell surface. Interfering with MT1-MMP-tetraspanin collaboration may be a useful therapeutic approach to limit cancer cell invasion and metastasis.


Assuntos
Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , Estabilidade Enzimática , Fibronectinas/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Ligação Proteica , Transporte Proteico , Frações Subcelulares/enzimologia
7.
Neoplasia ; 11(1): 77-86, 4p following 86, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19107234

RESUMO

EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP), which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP) known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.


Assuntos
Antígenos CD/fisiologia , Genes Supressores de Tumor/fisiologia , Glioblastoma/genética , Proteínas de Membrana/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Células HeLa , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Imunoglobulinas/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Ligação Proteica , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Matrix Biol ; 26(4): 291-305, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17175151

RESUMO

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.


Assuntos
Neoplasias da Mama/metabolismo , Colágeno Tipo I/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Integrina alfaVbeta3/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ativação Enzimática , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias
9.
J Biol Chem ; 281(10): 6826-40, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16407177

RESUMO

Type I collagen (Col I)-stimulated matrix metalloproteinase-2 (MMP-2) activation via membrane type 1 MMP (MT1-MMP) involves both a transcriptional increase in MT1-MMP expression and a nontranscriptional response mediated by preexisting MT1-MMP. In order to identify which MT1-MMP domains were required for the nontranscriptional response, MCF-7 cells that lack endogenous MT1-MMP were transfected with either wild type or domain mutant MT1-MMP constructs. We observed that mutant constructs lacking the MT1-MMP cytoplasmic tail were able to activate MMP-2 in response to Col I but not a construct lacking the MT1-MMP hemopexin domain. Col I did not alter total MT1-MMP protein levels; nor did it appear to directly induce MT1-MMP oligomerization. Col I did, however, redistribute preexisting MT1-MMP to the cell periphery compared with unstimulated cells that displayed a more diffuse staining pattern. In addition, Col I blocked the internalization of MT1-MMP in a dynamin-dependent manner via clathrin-coated pit-mediated endocytosis. This mechanism of impaired internalization is different from that reported for concanavalin A, since it is not mediated by the cytoplasmic tail of MT1-MMP but rather by the hemopexin domain. In summary, upon Col I binding to its cell surface receptor, MT1-MMP internalization via clathrin-coated pit-mediated endocytosis is impaired through interactions with the hemopexin domain, thereby regulating its function and ability to activate MMP-2.


Assuntos
Clatrina/fisiologia , Colágeno Tipo I/fisiologia , Metaloproteinase 1 da Matriz/metabolismo , Linhagem Celular Tumoral , Concanavalina A/metabolismo , Endocitose/fisiologia , Hemopexina/genética , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
Int J Cancer ; 114(4): 544-54, 2005 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-15551360

RESUMO

In human breast cancer (HBC), as with many carcinoma systems, most matrix metalloproteinases (MMPs) are largely expressed by the stromal cells, whereas the tumour cells are relatively silent in MMP expression. To determine the tissue source of the most relevant MMPs, we xenografted HBC cell lines and HBC tissues into the mammary fat pad (MFP) or bone of immunocompromised mice and measured the expression of human and mouse MMP-2, -9, -11, -13, membrane-type-1 MMP (MT1-MMP), MT2-MMP and MT3-MMP by species-specific real-time quantitative RT-PCR. Our data confirm a stromal origin for most tumour-associated MMPs and indicate marked and consistent upregulation of stromal (mouse) MMP-13 and MT1-MMP in all xenografts studied, irrespective of implantation in the MFP or bone environments. In addition, we show increased expression of both human MMP-13 and human MT1-MMP by the MDA-MB-231 tumour cells grown in the MFP compared to in vitro production. MMP protein and activity data confirm the upregulation of MMP mRNA production and indicate an increase in the activated MMP-2 species as a result of tumour implantation. These data directly demonstrate tumour induction of MMP production by stromal cells in both the MFP and bone environments. These xenografts are a valuable means for examining in vivo production of MMPs and suggest that MMP-13 and MT1-MMP will be relevant targets for inhibiting breast cancer progression.


Assuntos
Neoplasias da Mama/patologia , Colagenases/biossíntese , Colagenases/metabolismo , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Primers do DNA/química , DNA Complementar/metabolismo , Progressão da Doença , Feminino , Humanos , Metaloproteinase 11 da Matriz , Metaloproteinase 13 da Matriz , Metaloproteinase 14 da Matriz , Metaloproteinase 15 da Matriz , Metaloproteinase 16 da Matriz , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/biossíntese , Metaloendopeptidases/metabolismo , Metalotioneína 3 , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Transplante de Neoplasias , Reação em Cadeia da Polimerase , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
11.
FEBS Lett ; 553(3): 457-63, 2003 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-14572669

RESUMO

A tissue inhibitor of metalloproteinases-2 (TIMP-2)-independent mechanism for generating the first activational cleavage of pro-matrix metalloproteinase-2 (MMP-2) was identified in membrane type-1 MMP (MT1-MMP)-transfected MCF-7 cells and confirmed in TIMP-2-deficient fibroblasts. In contrast, the second MMP-2-activational step was found to be TIMP-2 dependent in both systems. MMP-2 hemopexin C-terminal domain was found to be critical for the first step processing, confirming a need for membrane tethering. We propose that the intermediate species of MMP-2 forms the well-established trimolecular complex (MT1-MMP/TIMP-2/MMP-2) for further TIMP-2-dependent autocatalytic cleavage to the fully active species. This alternate mechanism may supplement the traditional TIMP-2-mediated first step mechanism.


Assuntos
Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Animais , Sítios de Ligação , Neoplasias da Mama/metabolismo , Domínio Catalítico , Linhagem Celular , Ativação Enzimática , Precursores Enzimáticos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Gelatinases/química , Humanos , Metaloproteinase 14 da Matriz , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/química , Metaloendopeptidases/genética , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidor Tecidual de Metaloproteinase-2/química , Inibidor Tecidual de Metaloproteinase-2/genética , Transfecção , Células Tumorais Cultivadas
12.
Expert Rev Mol Med ; 5(23): 1-39, 2003 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-14585170

RESUMO

Angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is an integral part of physiological processes such as embryonic development, the female reproductive cycle and wound healing. Angiogenesis is also central to a variety of pathologies including cancer, where it is recognised as being crucial for the growth of solid tumours. Matrix metalloproteinases (MMPs) are a family of soluble and membrane-anchored proteolytic enzymes that can degrade components of the extracellular matrix (ECM) as well as a growing number of modulators of cell function. Several of the MMPs, most notably MMP-2 and -9 and membrane-type-1 MMP (MT1-MMP), have been linked to angiogenesis. Potential roles for these proteases during the angiogenic process include degradation of the basement membrane and perivascular ECM components, liberation of angiogenic factors, production of endogenous angiogenic inhibitors, and the unmasking of cryptic biologically relevant sites in ECM components. This review brings together what is currently known about the functions of the MMPs and the closely related adamalysin metalloproteinase (ADAM) family in angiogenesis, and discusses how this information might be useful in manipulation of the angiogenic process, with a view to controlling aberrant neovascularisation.


Assuntos
Metaloproteases/metabolismo , Neovascularização Patológica/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/patologia , Vasos Sanguíneos/fisiologia , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos , Humanos , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Cicatrização
13.
J Cell Sci ; 115(Pt 17): 3427-38, 2002 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-12154073

RESUMO

Macro- and microvascular endothelial cells (EC) formed tubular structures when cultured within a 3D fibrin matrix, a process that was enhanced by vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), hepatocyte growth factor/scatter factor (HGF/SF) and an angiogenic cocktail composed of nine angiogenic factors. Endothelial tubulogenesis was also increased in co-culture with tumour cells such as U87 glioma cells, but not with non-tumorigenic cell types such as Madin-Darby canine kidney (MDCK) epithelial cells. VEGF/FGF-2-stimulated tube formation was dependent on metalloproteinase function [it is inhibited by the addition of tissue inhibitor of metalloproteinases-2 (TIMP-2)], whereas aprotinin, E64 [trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane] and pepstatin had no effect. In addition, TIMP-4 also inhibited tubulogenesis, but TIMP-1 or the C-terminal haemopexin domain of matrix metalloproteinase-2 (MMP-2) (PEX) and an anti-MMP-2 function-blocking antibody were unable to block tube formation. This suggests that MMP-2 and other soluble MMPs are not essential for tubulogenesis in fibrin gels, instead TIMP-1-insensitive MMPs, such as members of the membrane type-MMPs (MT-MMP) sub-group (MT1-, MT2-, MT3- or MT5-MMP), are required for this process. Further support for a role for MT1-MMP in endothelial tubulogenesis is that recombinant Y36G N-terminal TIMP-2 mutant protein, which retains an essentially unaltered apparent inhibition constant (K(i)(app)) for several MMPs compared to wild-type N-TIMP-2 but is a 40-fold poorer inhibitor of MT1-MMP, was unable to block tubulogenesis. Furthermore, when EC were cultured within fibrin gels, the mRNA levels of several MMPs (including MT1-MMP, MT2-MMP, MT3-MMP and MMP-2) increased during tubulogenesis. Therefore MT-MMPs and specifically MT1-MMP are likely candidates for involvement during endothelial tubulogenesis within a fibrin matrix, and thus their blockade may be a viable strategy for inhibition of angiogenesis.


Assuntos
Endotélio Vascular/crescimento & desenvolvimento , Fibrina/metabolismo , Metaloendopeptidases/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Géis , Substâncias de Crescimento/farmacologia , Humanos , Metaloproteinase 15 da Matriz , Metaloproteinase 16 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/metabolismo , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo
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