The suitable experimental animal model is important in research of pathogenesis and therapeutic strategies of diabetic foot ulcer, and the murine model is the most commonly used one at present. It can be divided into two types: the animal model simulating pathological conditions and the model simulating clinical symptoms. This article reviews the current research progress on the mechanisms of diabetic ulcer pathogenesis, and relevant treatment strategies, including the inhibition of matrix metalloproteinases (MMPs) expression, promotion of angiogenesis and anti-inflammatory therapy.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Diabetic Foot ; etiology ; genetics ; therapy ; Disease Models, Animal ; Humans ; Matrix Metalloproteinase Inhibitors ; therapeutic use ; Matrix Metalloproteinases ; genetics ; metabolism ; Mice ; Neovascularization, Physiologic ; physiology

PURPOSE: To determine the effect of exogenous nitric oxide (NO) on the migration of trabecular meshwork (TM) cells and its association with expression of matrix metalloproteinases (MMPs). METHODS: Primary human TM cells treated with 1 or 10 microM S-nitroso-N-acetyl-penicillamine (SNAP) and examined for changes in adherence. TM cells were seeded onto transwell culture inserts, and changes in their migratory activity were quantified. Reverse transcription polymerase chain reaction was performed to determine the relative changes in mRNA expression of MMPs and tissue inhibitor of metalloproteinases (TIMPs). RESULTS: Treatment with SNAP did not significantly suppress TM cell adhesion or migration (p > 0.05). Treatment of TM cells with 10 microM SNAP decreased expression of MMP-2 and increased expression of membrane type MMP-1 and TIMP-2. Treatment with interleukin-1alpha triggered MMP-3 expression but did not exert significant effects on MMP-3 activation in response to SNAP. CONCLUSIONS: These data suggest that NO revealed no significant effect on the migration of TM cells because NO decreased MMP-2 and increased TIMP-2 expression. Although expression of certain MMPs and TIMPs change in response to NO donors, NO may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of TM cells.
Cell Movement/*drug effects ; Cell Survival/drug effects ; Cells, Cultured ; DNA Primers/chemistry ; Gene Expression Regulation, Enzymologic/*physiology ; Humans ; Matrix Metalloproteinases/*genetics ; Nitric Oxide Donors/*pharmacology ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; S-Nitroso-N-Acetylpenicillamine/*pharmacology ; Tissue Inhibitor of Metalloproteinase-2/*genetics ; Trabecular Meshwork/cytology/*drug effects/enzymology

The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1β (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Chondrocytes ; cytology ; enzymology ; Gene Silencing ; Lentivirus ; genetics ; Matrix Metalloproteinases ; metabolism ; Rabbits ; Urokinase-Type Plasminogen Activator ; genetics

OBJECTIVETo investigate the effects of adenovirus-delivered angiopoietin-1 siRNA (Ad. Ang-1siRNA) on the expression of matrix metalloproteinase-2, 9 (MMP-2, 9) and tissue inhibitor of metallopro-teinase-1 (TIMP-1) in rats with acute lung injury (ALI) induced by phosgene (Psg).

METHODSWe first established a rat model of Psg-induced acute lung injury (ALI). The rats were randomly divided into 6 groups: air control group with exposure to air, air+adenovirus (air+Ad) group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after air exposure, air+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after air exposure, Psg group with exposure to 8.33 mg/L Psg (purity 100%, of the same volume as the inhaled air in the air control group) for 5 min, Psg+Ad group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after exposure to the same dose of Psg, and Psg+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after exposure to the same dose of Psg. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected 36 h after exposure. The protein expression of Ang-1, MMP-2, 9, and TIMP-1 in serum and BALF was determined by double-antibody sandwich ELISA. RT-PCR was used to determine the mRNA levels of Ang-1, MMP-2, 9, and TIMP-1 in lung tissue. The protein expression of MMP-2, 9 and TIMP-1 in lung tissue was determined by Western blot.

RESULTSA rat model of Psg-induced ALI was successfully established. The levels of MMP-2, 9 in serum, BALF, and lung tissue were significantly increased in the Psg group and Psg+Ad/Ang1 group as compared with the control group (P<0.01); no significant change was observed in serum TIMP-1 protein expression (P>0.05); interestingly, TIMP-1 protein expression in BALF and lung tissue was significantly increased (P<0.01). Compared with the Psg group, the Psg+Ad/Ang1 group showed a significant decrease in MMP-2, 9 expression in BALF, serum, and lung tissue (P<0.05), but no significant change in protein expression of TIMP-1 was discovered (P>0.05).

CONCLUSIONAd.Ang-1siRNA has a potential beneficial effect in rats with Psg-induced ALI through inhibition of MMP-2, 9 expression, but has no significant effect on the expression of TIMP-1.

Acute Lung Injury ; chemically induced ; metabolism ; Adenoviridae ; genetics ; Angiopoietin-1 ; physiology ; Animals ; Bronchoalveolar Lavage Fluid ; Chemical Warfare Agents ; toxicity ; Disease Models, Animal ; Lung ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; Matrix Metalloproteinases ; metabolism ; Phosgene ; toxicity ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

OBJECTIVEThe aim of this study was to determine whether inhibition of cyclophilin A by lentivirus-mediated RNA interference (RNAi) could inhibit progression of atherosclerotic plaques and increase collagen production.

METHODSAtherosclerostic plaque model was induced by rapid perivascular carotid silicone collar placement in ApoE(-/-) mice. The recombinant CyPA-RNAi-Lentivirus (CyPA-RNAi-LV) or negative control-green fluorescent protein-Lentivirus (NC-GFP-LV) were constructed and transfected into right carotid plaques, respectively. Using the local injection method, ApoE(-/-) mice carotid artery plaque were intervened 10 min in the silicone collar placement with 10 µl (1.0 × 10⁸ TU/ml) lentivirus vector. The areas and CyPA expression of plaques were analyzed by morphological observation, real-time polymerase chain reaction (RT-PCR) and Western blot respectively.

RESULTSCyPA-RNAi-LV not only prevented plaques progression ((9 085 ± 671) µm² to (18 021 ± 1 901) µm²), but also decreased plaque lipid content ((28.9 ± 6.3)% to (17.8 ± 4.5)%), increased plaque collagen content ((24.2 ± 4.8)% to (35.1 ± 5.2)%) at 6 weeks after lentivirus transfection. The intima/media ratio (0.36 ± 0.11 vs. 0.65 ± 0.12, P < 0.05) and degree of lumen stenosis (intima/lumen ratios, 0.18 ± 0.02 vs. 0.33 ± 0.03, P < 0.05) were also significantly reduced by CyPA-RNAi-LV. Moreover, RT-PCR analysis revealed downregulated expressions of proinflammatory cytokines and matrix metalloproteinases (MMP-9 -17.5%) in the CyPA-RNAi-LV group.

CONCLUSIONLentivirus-mediated CyPA silencing by siRNA could inhibit plaques progression and reduce local inflammation through the anti-inflammatory effects in this model.

Animals ; Apolipoproteins E ; Cyclophilin A ; biosynthesis ; genetics ; Disease Progression ; Gene Silencing ; Genetic Vectors ; Inflammation ; Lentivirus ; Matrix Metalloproteinase 9 ; Matrix Metalloproteinases ; Mice ; Plaque, Atherosclerotic ; RNA Interference ; RNA, Small Interfering ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

BACKGROUNDReceptor interacting protein 1 (RIP1), which plays a key role in apoptosis, cell survival and programmed cell necrosis, is one of the most important proteins in the RIP family. The purpose of this study was to investigate the roles of RIP1 in the apoptosis, the generation of reactive oxygen species (ROS) and the expression of matrix metalloproteinases (MMPs) induced by ultraviolet B (UVB) in fibroblasts.

METHODSsiRNA targeting RIP1 was used to silence RIP1 expression in the NIH3T3 fibroblasts. The mRNA and protein levels of MMP-1 and MMP-3, caspase-3 and -8 activities, and ROS activities were determined by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), immunoblotting, caspase activity assay, immunofluorescence, and flow cytometry.

RESULTSThe mRNA and protein expressions of MMP-1 and MMP-3 were significantly increased in RIP1 deficient NIH3T3 cells at 24 hours after UVB treatment. At 24 hours after exposure to UVB, RIP1 deficient NIH3T3 cells presented apoptotic morphology, and the apoptosis rate was significantly increased accompanied by pronounced increase in caspase-8 and -3 activities. ROS production was inhibited by UVB at 12 hours in RIP1 deficient NIH3T3 cells.

CONCLUSIONRIP1 is involved in NIH3T3 cell damage induced by UVB via participating in the apoptosis, expression of MMPs and ROS production.

Animals ; Apoptosis ; GTPase-Activating Proteins ; genetics ; metabolism ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Matrix Metalloproteinase 3 ; genetics ; metabolism ; Matrix Metalloproteinases ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; RNA, Small Interfering ; Reactive Oxygen Species ; metabolism ; Ultraviolet Rays

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.
Adult ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/*diagnostic use/genetics/isolation & purification ; Central Nervous System Parasitic Infections/*diagnosis/parasitology ; Gnathostoma/enzymology/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/parasitology ; Healthy Volunteers ; Humans ; Immunoblotting/methods ; Immunoglobulin G/blood ; Matrix Metalloproteinases/*diagnostic use/genetics/isolation & purification ; Parasitology/*methods ; Prospective Studies ; Recombinant Proteins/diagnostic use/genetics/isolation & purification ; Sensitivity and Specificity ; Serologic Tests/methods ; Thailand

This paper is aimed to investigate the effect of titanium (Ti) particles and tumor necrosis factor alpha (TNF-alpha) on the expressions of MMP-1, 2, 3 in human synovial cells, so as to explore the possible mechanism of osteolysis post-operation of metal-on-metal total joint arthroplasty in human synovial cells induced by Ti particles. In vitro cell cultures, human synovial cells were treated by Ti particles and/or TNF-alpha. The total RNA was isolated at 2 hours after the treatment. The gene expression of MMP-1, 2, 3 was analyzed by Semi-quantitative Reverse-transcriptional PCR and quantitative real-time PCR. Cell supernatant was collected at 12, 24, 48 hours after the treatment and Gelatin zymography was performed to detect the activity of MMP-2. Compared to those in the control group (untreated), Ti particles and TNF-alpha increased the gene expression of MMP-1, 2, 3 respectively (P < 0.05), and the effect of combination of the two was even more significant (P < 0.01). The trend of activities of MMP-2 is similar with gene expression. Ti particles and TNF-alpha increased MMP-2 activities by 1.3 times and 1.5 times respectively (P < 0.05), and the combination of the two increased by 1.7 times (P < 0.01). Ti particles and TNF-alpha-induced the stimulation of MMP-1, 2, 3 expressions and MMP-2 activities in human knee joint synovial cells may be involved in aseptic loosening after metal-on-metal arthroplasty through increasing the degradation of bone matrix and declining of osseous support structure mechanics.
Cells, Cultured ; Humans ; Joint Prosthesis ; Knee Joint ; cytology ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 3 ; genetics ; metabolism ; Matrix Metalloproteinases ; genetics ; metabolism ; Particle Size ; Prosthesis Failure ; adverse effects ; RNA ; genetics ; metabolism ; Synovial Membrane ; cytology ; enzymology ; Titanium ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

The progress of research on the the anterior cruciate ligament (ACL) wound healing demonstrates that the synovial tissue in the knee joint plays a very important role in the healing process of injured ACL. Therefore, the molecular response mechanisms of lysyl oxidase (LOX) and matrix metalloproteina (MMP) in normal/injured ACL fibroblast cells could be considered to perform the major analysis function of injured ACL healing mechanism. The mRNA expressions of LOXs and MMPs and the activity expressions of MMP-2 in ACL fibroblasts co-cultured with synovial cells were analyzed by quantitative real-time PCR and zymography. The results showed that co-culture could regulate the mRNA expressions of LOXs and MMPs in the ACL fibroblasts cells. These results suggest that the differential expressions of LOXs and MMP-1, 2, 3 in co-cultured ACL indicate that interaction crosstalk do exist between ACL cells and synovial cells and provide a theoretical basis for subsequent exploration of the mechanisms and treatment of ACL injury and repair.
Anterior Cruciate Ligament ; cytology ; Anterior Cruciate Ligament Injuries ; Coculture Techniques ; Fibroblasts ; cytology ; metabolism ; Humans ; Knee Injuries ; physiopathology ; Knee Joint ; cytology ; Matrix Metalloproteinases ; genetics ; metabolism ; Protein-Lysine 6-Oxidase ; genetics ; metabolism ; Synovial Membrane ; cytology ; Wound Healing ; physiology

The purpose of this study was to investigate the repair of the osteoarthritis(OA)-induced cartilage injury by transfecting the new TGF-β3 fusion protein (LAP-MMP-mTGF-β3) with targeted therapy function into the bone marrow-derived mesenchymal stem cells (MSCs) in rats. The recombinant of pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, so as to construct the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3. pIRES-EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cultured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction (ACLT). The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Green and graded by Mankin's scale. pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein (39 kD) was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.
Animals ; Base Sequence ; Blotting, Western ; Bone Marrow Cells ; metabolism ; Cartilage, Articular ; pathology ; surgery ; Cell Differentiation ; genetics ; Cells, Cultured ; Chondrocytes ; metabolism ; Chondrogenesis ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Matrix Metalloproteinases ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Microscopy, Fluorescence ; Molecular Sequence Data ; Osteoarthritis ; surgery ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Transforming Growth Factor beta3 ; genetics ; metabolism ; Treatment Outcome