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2.
Data Brief ; 8: 230-4, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27331093

ABSTRACT

The data described herein are related to the article entitled "Tumor necrosis factor-α regulates triggering receptor expressed on myeloid cells-1-dependent matrix metalloproteinases in the carotid plaques of symptomatic patients with carotid stenosis" (Rao et al., 2016) [1]. Additional data are provided on the dose-response effect of TNF-α, TREM-1 antibody and recombinant rTREM-1/Fc fusion chimera (TREM-1/FC) on the expression of MMP-1 and MMP-9 in vascular smooth muscle cells (VSMCs) isolated from human carotid endarterectomy tissues. Data are also presented on the distribution of CD86+ M1- and CD206+ M2-macrophages and their co-localization with TREM-1 in symptomatic carotid plaques as visualized by dual immunofluorescence. The interpretation of this data and further extensive insights can be found in Rao et al. (2016) [1].

3.
PLoS One ; 11(5): e0154802, 2016.
Article in English | MEDLINE | ID: mdl-27148736

ABSTRACT

Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory cells, including T-lymphocytes, dendritic cells (DCs) and macrophages that are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. In atherosclerotic tissue the number of DCs increases with the progression of the lesion and produce several inflammatory cytokines and growth factors. Triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role in inflammation. However, relationship of DCs and the role of TREM-1 with the stability of atherosclerotic plaques have not been examined. In this study, we investigated the heterogeneity of the plaque DCs, myeloid (mDC1 and mDC2) and plasmacytoid (pDCs), and examined the expression of TREM-1 and their co-localization with DCs in the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. We found increased expression of HLA-DR, fascin, and TREM-1 and decreased expression of TREM-2 and α-smooth muscle actin in S compared to AS atherosclerotic carotid plaques. Both TREM-1 and fascin were co-localized suggesting increased expression of TREM-1 in plaque DCs of S compared to AS patients. These data were supported by increased mRNA transcripts of TREM-1 and decreased mRNA transcripts of TREM-2 in carotid plaques of S compared to AS patients. There was higher density of both CD1c+ mDC1 and CD141+ mDC2 in the carotid plaques from AS compared to S patients, where as the density of CD303+ pDCs were higher in the carotid plaques of S compared to AS patients. These findings suggest a potential role of pDCs and TREM-1 in atherosclerotic plaque vulnerability. Thus, newer therapies could be developed to selectively block TREM-1 for stabilizing atherosclerotic plaques.


Subject(s)
Carotid Stenosis/immunology , Dendritic Cells/immunology , Actins/metabolism , Carotid Stenosis/metabolism , Carotid Stenosis/surgery , Case-Control Studies , Endarterectomy, Carotid , Female , HLA-DR Antigens/metabolism , Humans , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Plaque, Atherosclerotic/pathology , RNA, Messenger/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Triggering Receptor Expressed on Myeloid Cells-1
4.
Atherosclerosis ; 248: 160-9, 2016 May.
Article in English | MEDLINE | ID: mdl-27017522

ABSTRACT

OBJECTIVE: To determine the relationship between increased triggering receptor expressed on myeloid cells (TREM)-1 and plaque stability in atherosclerotic carotid stenosis. METHODS: The mRNA transcripts and protein for TREM-1, MMP-1, MMP-9, collagen type I (COL1A1) and collagen type III (COL3A1) were analyzed by qPCR and immunofluorescence in both tissues and VSMCs isolated from atherosclerotic carotid plaques of symptomatic and asymptomatic patients with carotid stenosis. RESULTS: The TREM-1, MMP-1 and MMP-9 mRNA transcripts were significantly increased (TREM-1, p < 0.01; MMP-1, p < 0.01 and MMP-9, p < 0.001) while COL1A1 and COL3A1 mRNA transcripts were decreased (p < 0.001) in VSMCs isolated from carotid plaques of symptomatic (S) than asymptomatic (AS) patients. Stimulation of cells with TNF-α further increased the mRNA transcripts of TREM-1, MMPs, COL1A1 and COL3A1. Modulation of TREM-1 by treatment with TREM-1 decoy receptor rTREM-1/Fc, and either TREM-1 antibodies or TREM-1 siRNA attenuated the TNF-α-induced expression of MMP-1 and MMP-9 (p < 0.01) and COL1A1 and COL3A1 (p < 0.01) in S compared to AS VSMCs isolated from carotid plaques. Inhibition of NF-kB (BAY 11-7085), JNK (SP600125) and PI3K (LY294002) signaling pathways decreased the expression of TREM-1 (p < 0.01), MMP-1 (p < 0.001) and MMP-9 (p < 0.01) in TNF-α-treated VSMCs isolated from S carotid plaques compared to AS patients. CONCLUSION: Increased expression of TREM-1 in S compared to AS patients involving MMP-1 and MMP-9 suggest a potential role of TREM-1 in plaque destabilization. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.


Subject(s)
Carotid Stenosis/metabolism , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 9/metabolism , Membrane Glycoproteins/metabolism , Plaque, Atherosclerotic/metabolism , Receptors, Immunologic/metabolism , Tumor Necrosis Factor-alpha/metabolism , Aged , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Collagen Type III/metabolism , Cytokines/metabolism , Female , Humans , Male , Microscopy, Fluorescence , Middle Aged , Muscle, Smooth, Vascular/cytology , RNA, Small Interfering/metabolism , Signal Transduction , Triggering Receptor Expressed on Myeloid Cells-1
5.
Am J Physiol Heart Circ Physiol ; 309(6): H1075-86, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26254334

ABSTRACT

Although degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be involved in symptomatic (S) carotid plaques in atherosclerosis, the mechanisms of MMP expression are poorly understood. Here, we demonstrate that collagen loss in vascular smooth vessel cells (VSMCs) isolated from S plaques was induced by epidermal growth factor (EGF) through the activation of p38-MAPK and JNK-MAPK pathways. Inhibitors of p38-MAPK and JNK-MAPK signaling pathways downregulated the expression of MMP-1 and MMP-9. In addition, we examined whether v-ets erythroblastosis virus E26 oncogene homologue 1 (Ets-1), an important regulator of different genes, is involved in destabilizing S plaques in patients with carotid stenosis. We demonstrate that EGF induces Ets-1 expression and decreases interstitial and basement membrane collagen in vascular smooth muscle cells (VSMCs) from patients with carotid stenosis. Increased expression of MMP-1 and -9 and decreased collagen mRNA transcripts were also found in Ets-1-overexpressed VSMCs. Transfection with both dominant-negative form of Ets-1 and small interfering RNA blocked EGF-induced MMP-1 and -9 expressions and increased the mRNA transcripts for collagen I (α1) and collagen III (α1) in S compared with asymptomatic (AS) carotid plaques. Inhibitors of p38-MAPK (SB202190) and JNK-MAPK (SP600125) signaling pathways decreased the expression of Ets-1, MMP-1, and MMP-9 and increased collagen type I and III expression in EGF-treated VSMCs. This study provides a mechanistic insight into the role of Ets-1 in the plaque destabilization in patients with carotid stenosis involving p38-MAPK and JNK signaling pathways.


Subject(s)
Carotid Stenosis/genetics , Epidermal Growth Factor/genetics , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Proto-Oncogene Protein c-ets-1/genetics , RNA, Messenger/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , Aged , Carotid Stenosis/metabolism , Cell Culture Techniques , Collagen/genetics , Collagen/metabolism , Epidermal Growth Factor/metabolism , Female , Gene Expression Regulation , Humans , MAP Kinase Signaling System , Male , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Middle Aged , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
6.
Mol Carcinog ; 54(10): 1026-36, 2015 Oct.
Article in English | MEDLINE | ID: mdl-24798404

ABSTRACT

Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness.


Subject(s)
ADAM Proteins/genetics , Gene Expression/genetics , Receptor, ErbB-2/genetics , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Up-Regulation/genetics , ADAM12 Protein , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line , Cell Line, Tumor , DNA Damage/genetics , Disease Progression , Humans , Mice , Mice, Transgenic , Skin/pathology , Ultraviolet Rays/adverse effects
7.
Physiol Rep ; 2(2): e00224, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-24744893

ABSTRACT

Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)-mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP-9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP-9, MMP-1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP-9 activity and mRNA transcripts for MMP-9, MMP-1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP-9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF-treated VSMCs of both groups. Additionally, the magnitude in decreased MMP-9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP-9 gene with siRNA in EGF-treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP-9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.

8.
Kidney Int ; 76(9): 968-76, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19710627

ABSTRACT

Patients with Alport's syndrome develop a number of pro-inflammatory cytokine and matrix metalloproteinase (MMP) abnormalities that contribute to progressive renal failure. Changes in the composition and structure of the glomerular basement membranes likely alter the biomechanics of cell adhesion and signaling in these patients. To test if enhanced strain on the capillary tuft due to these structural changes contributes to altered gene regulation, we subjected cultured podocytes to cyclic biomechanical strain. There was robust induction of interleukin (IL)-6, along with MMP-3, -9, -10, and -14, but not MMP-2 or -12 by increased strain. Neutralizing antibodies against IL-6 attenuated the strain-mediated induction of MMP-3 and -10. Alport mice treated with a general inhibitor of nitric oxide synthase (L-NAME) developed significant hypertension and increased IL-6 and MMP-3 and -10 in their glomeruli relative to those of normotensive Alport mice. These hypertensive Alport mice also had elevated proteinuria along with more advanced histological and ultrastructural glomerular basement membrane damage. We suggest that MMP and cytokine dysregulation may constitute a maladaptive response to biomechanical strain in the podocytes of Alport patients, thus contributing to glomerular disease initiation and progression.


Subject(s)
Glomerular Basement Membrane/metabolism , Interleukin-6/genetics , Kidney Glomerulus/metabolism , Matrix Metalloproteinases/genetics , Nephritis, Hereditary/genetics , Podocytes/metabolism , Adaptation, Physiological/genetics , Animals , Blood Pressure , Cells, Cultured , Cytoskeleton/metabolism , Disease Models, Animal , Gene Expression Regulation , Hypertension/chemically induced , Hypertension/genetics , Hypertension/physiopathology , Interleukin-6/metabolism , Kidney Glomerulus/physiopathology , Matrix Metalloproteinase 10/genetics , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred C57BL , NG-Nitroarginine Methyl Ester , Nephritis, Hereditary/metabolism , Nephritis, Hereditary/physiopathology , Proteinuria/chemically induced , Proteinuria/genetics , Proteinuria/physiopathology , RNA, Messenger/metabolism , Sodium Chloride, Dietary , Stress, Mechanical , Time Factors
9.
Am J Pathol ; 172(3): 761-73, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18258846

ABSTRACT

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.


Subject(s)
Gene Expression Regulation, Enzymologic , Integrin alpha1beta1/physiology , Matrix Metalloproteinases/genetics , Mesangial Cells/enzymology , Nephritis, Hereditary/genetics , p38 Mitogen-Activated Protein Kinases/physiology , Animals , Autoantigens/genetics , Biphenyl Compounds , Cells, Cultured , Collagen Type IV/genetics , Disease Models, Animal , Gene Expression Regulation, Enzymologic/drug effects , Integrin alpha1beta1/genetics , Matrix Metalloproteinases/metabolism , Mesangial Cells/metabolism , Mice , Mice, Knockout , Nephritis, Hereditary/enzymology , Nephritis, Hereditary/pathology , Organic Chemicals/pharmacology , Phenylbutyrates , Tissue Inhibitor of Metalloproteinases/genetics , Tissue Inhibitor of Metalloproteinases/metabolism
10.
Am J Pathol ; 169(1): 32-46, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16816359

ABSTRACT

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.


Subject(s)
Glomerular Basement Membrane/pathology , Metalloendopeptidases/metabolism , Nephritis, Hereditary/enzymology , Nephritis, Hereditary/pathology , Animals , Blotting, Northern , Blotting, Western , Enzyme Inhibitors/pharmacology , Glomerular Basement Membrane/drug effects , Humans , Immunohistochemistry , In Situ Hybridization , Matrix Metalloproteinase 12 , Metalloendopeptidases/drug effects , Mice , Microscopy, Electron, Transmission , Receptors, CCR2 , Receptors, Chemokine/drug effects , Receptors, Chemokine/metabolism , Reverse Transcriptase Polymerase Chain Reaction
11.
PLoS Med ; 3(4): e100, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16509766

ABSTRACT

BACKGROUND: Glomerular basement membrane (GBM), a key component of the blood-filtration apparatus in the in the kidney, is formed through assembly of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations or deletions involving alpha3(IV), alpha4(IV), or alpha5(IV) chains of type IV collagen in the GBM have been identified as the cause for Alport syndrome in humans, a progressive hereditary kidney disease associated with deafness. The pathological mechanisms by which such mutations lead to eventual kidney failure are not completely understood. METHODS AND FINDINGS: We showed that increased susceptibility of defective human Alport GBM to proteolytic degradation is mediated by three different matrix metalloproteinases (MMPs)--MMP-2, MMP-3, and MMP-9--which influence the progression of renal dysfunction in alpha3(IV)-/- mice, a model for human Alport syndrome. Genetic ablation of either MMP-2 or MMP-9, or both MMP-2 and MMP-9, led to compensatory up-regulation of other MMPs in the kidney glomerulus. Pharmacological ablation of enzymatic activity associated with multiple GBM-degrading MMPs, before the onset of proteinuria or GBM structural defects in the alpha3(IV)-/- mice, led to significant attenuation in disease progression associated with delayed proteinuria and marked extension in survival. In contrast, inhibition of MMPs after induction of proteinuria led to acceleration of disease associated with extensive interstitial fibrosis and early death of alpha3(IV)-/- mice. CONCLUSIONS: These results suggest that preserving GBM/extracellular matrix integrity before the onset of proteinuria leads to significant disease protection, but if this window of opportunity is lost, MMP-inhibition at the later stages of Alport disease leads to accelerated glomerular and interstitial fibrosis. Our findings identify a crucial dual role for MMPs in the progression of Alport disease in alpha3(IV)-/- mice, with an early pathogenic function and a later protective action. Hence, we propose possible use of MMP-inhibitors as disease-preventive drugs for patients with Alport syndrome with identified genetic defects, before the onset of proteinuria.


Subject(s)
Genetic Predisposition to Disease , Glomerular Basement Membrane/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 9/metabolism , Nephritis, Hereditary/genetics , Nephritis, Hereditary/physiopathology , Animals , Autoantigens/genetics , Autoantigens/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Disease Progression , Extracellular Matrix/metabolism , Glomerular Basement Membrane/physiology , Humans , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Proteinuria/physiopathology , Substrate Specificity , Survival Analysis , Up-Regulation
12.
J Biol Chem ; 280(23): 21882-92, 2005 Jun 10.
Article in English | MEDLINE | ID: mdl-15824107

ABSTRACT

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.


Subject(s)
Brain Neoplasms/metabolism , Gene Expression Regulation, Enzymologic , Glioma/metabolism , Matrix Metalloproteinase 9/metabolism , Neovascularization, Pathologic , RNA Interference , RNA, Double-Stranded/genetics , Receptors, Cell Surface/metabolism , Animals , Brain Neoplasms/blood supply , Brain Neoplasms/pathology , Cell Line, Transformed , Cell Line, Tumor , Cell Proliferation , Collagen/pharmacology , Down-Regulation , Drug Combinations , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gene Silencing , Genetic Vectors , Glioblastoma/metabolism , Glioma/blood supply , Glioma/therapy , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry , Laminin/pharmacology , MAP Kinase Signaling System , Male , Mice , Mice, Nude , Models, Biological , Models, Genetic , Neoplasm Invasiveness , Nucleic Acid Conformation , Phosphorylation , Promoter Regions, Genetic , Protein Structure, Tertiary , Proteoglycans/pharmacology , RNA, Small Interfering/metabolism , Receptors, Urokinase Plasminogen Activator , Time Factors , Transfection
13.
Am J Pathol ; 166(5): 1465-74, 2005 May.
Article in English | MEDLINE | ID: mdl-15855646

ABSTRACT

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.


Subject(s)
Matrix Metalloproteinases/metabolism , Nephritis, Hereditary/enzymology , Stria Vascularis/enzymology , Animals , Basement Membrane/pathology , Capillaries/drug effects , Capillaries/pathology , Computer Systems , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/genetics , Mice , Mice, Knockout , Nephritis, Hereditary/pathology , Protease Inhibitors/pharmacology , Pyrazines/pharmacology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stria Vascularis/drug effects , Sulfonamides/pharmacology
14.
Pediatr Nephrol ; 20(6): 732-9, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15782307

ABSTRACT

Matrix metalloproteinases (MMPs) play an important regulatory role in many biological and pathological processes and their specific role in Alport syndrome (AS) is not yet clearly defined. In this study, the naturally occurring canine X-linked AS was used to demonstrate a potential role for MMP-3 and MMP-7 in Alport renal pathogenesis. Recently, we demonstrated that the expression of MMP-2, MMP-9 and MMP-14 was upregulated in the renal cortex of dogs with a spontaneous form of XLAS. In the present study, we examined necropsy samples of renal cortex from normal and XLAS dogs for MMP-3 and MMP-7 as they have the potential to activate MMP-2 and MMP-9. Immunohistochemical analysis showed strong immunostaining for both MMP-3 and MMP-7 in the interstitial space of XLAS kidneys, while virtually no immunostaining was observed in similar fields from normal dogs. RT-PCR and casein zymography confirmed that both mRNA transcripts and activities of MMP-3 and MMP-7 are elevated in XLAS kidneys. The induction of these MMPs likely contributes to tissue destruction associated with the fibrogenic process, while augmenting the activation of MMP-2 and MMP-9 by MMP-3 and MMP-7 in XLAS. Thus, these data further implicate a role for the MMPs in progressive renal pathogenesis associated with AS.


Subject(s)
Dog Diseases/enzymology , Dog Diseases/genetics , Genetic Linkage , Kidney/enzymology , Matrix Metalloproteinases/metabolism , Nephritis, Hereditary/veterinary , X Chromosome , Animals , Dogs , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7/metabolism , Nephritis, Hereditary/genetics
15.
Kidney Int ; 63(5): 1736-48, 2003 May.
Article in English | MEDLINE | ID: mdl-12675849

ABSTRACT

BACKGROUND: Alport syndrome is a group of genetic disorders resulting from mutations in either the alpha3(IV), alpha4(IV) or alpha5(IV) collagen chains. The disease is characterized by a progressive glomerulonephritis, usually associated with a high-frequency specific sensorineural hearing loss, dot and fleck retinopathy, and lens abnormalities. Dogs with naturally occurring genetic disorders of basement membrane collagen (type IV) may serve as animal models of Alport syndrome. In this study, a well-characterized naturally occurring canine model was employed to demonstrate a potential role for matrix metalloproteinases (MMPs) in Alport renal disease pathogenesis. METHODS: Adolescent male dogs that developed renal failure were euthanized and necropsied. Clinicopathologic features of the disease were characterized, and kidneys from normal and Alport dogs were analyzed by gelatin zymography, Western blotting, in situ zymography, immunohistology, and by reverse transcription polymerase chain reaction (RT-PCR) for expression of MMP-2, MMP-9, and membrane type 1-MMP (MT1-MMP). RESULTS: Affected dogs developed proteinuria and rapidly progressive juvenile-onset chronic renal failure. The activities of MMP-2 and MMP-9 were significantly induced in Alport kidney. In situ zymography confirmed elevated active metalloproteinases in kidney cryosections of affected dogs. The mRNAs encoding MMP-2, MMP-9 and MT1-MMP were also increased in Alport dogs suggesting that elevated expression of MMPs reflects events in the progression of Alport syndrome in dogs. CONCLUSION: Elevated expression of MMP-2, MMP-9, and MT1-MMP is observed in fibrotic renal cortex from X-linked Alport syndrome dogs. These findings suggest that MMPs may play an important role in matrix accumulation associated with progressive renal scarring in this model.


Subject(s)
Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Nephritis, Hereditary/pathology , Nephritis, Hereditary/physiopathology , Animals , Dogs , Fibrosis , Gelatin , Gene Expression Regulation, Enzymologic , Kidney Cortex/enzymology , Kidney Cortex/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Nephritis, Hereditary/genetics , RNA, Messenger/analysis , X Chromosome
16.
Kidney Int ; 63(4): 1338-55, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12631350

ABSTRACT

BACKGROUND: In interstitial fibrosis, monocytes and myofibroblasts have been directly implicated in scarring, apoptosis, and tissue necrosis. While much has been done to explore the role of these cell types individually in fibrosis, the interactive dependency of monocytes and myofibroblasts has been only marginally explored. METHODS: Alport mice were treated or not with a soluble receptor inhibitor for transforming growth factor-beta 1 (TGF-beta 1), which was previously shown to inhibit the accumulation of myofibroblasts, but not monocytes, in the tubulointerstitium. Kidneys were examined for fibrosis using several matrix markers, TGF-beta 1 mRNA expression by in situ hybridization, apoptosis using the terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL) assay, expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) by dual immunofluorescence microscopy, MMP activity by gelatin and in situ zymography, MMP mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR), and basement membrane degradation by dual immunofluorescence confocal microscopy and electron microscopy. RESULTS: Treated mice showed a markedly reduced accumulation of matrix proteins. Tissue monocytes express TGF-beta 1 mRNA, and TGF-beta 1 is required for myofibroblast accumulation. The number of apoptotic cells was not influenced by TGF-beta 1 inhibition. Monocytes express MMP-2, MMP-9, TIMP-2, and TIMP-3. MMP activity and mRNA expression is equally up regulated in treated and untreated Alport mice. Tubular basement membranes (TBM) around clusters of monocytes are notably degraded. TGF-beta 1 inhibition does not extend the life of Alport mice. CONCLUSION: These studies demonstrate that monocytes may influence myofibroblast accumulation via TGF-beta1, and that monocytes, and not myofibroblasts, are associated with tubular atrophy in Alport mice. Elevated MMP expression and activity is associated with TBM destruction near monocytes clusters, suggesting an anoikis mechanism may contribute to apoptosis in this model.


Subject(s)
Apoptosis , Fibroblasts/pathology , Kidney/pathology , Monocytes/pathology , Nephritis, Hereditary/pathology , Animals , Atrophy , B-Lymphocytes/pathology , Fibrosis , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Nephritis, Hereditary/physiopathology , RNA, Messenger/analysis , T-Lymphocytes/pathology , Tissue Inhibitor of Metalloproteinases/genetics , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1
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