BACKGROUND AND OBJECTIVES: The octapeptide hormone of the renin-angiotensin system, angiotensin ii, regulates a wide variety of physiological responses including salt and water balance, blood pressure, and vascular tone. Contradictory results have been reported regarding the effects of angiotensin ii on vascular smooth mu-scle cell (VSMC) proliferation. The aim of the present study was to investigate the direct effect of angiotensin ii on the growth of VSMC. MATERIALS AND METHOD: Rat aortic smooth muscle cells were obtained by the combined collagenase and elastase methods. Cells between the 4th and 8th passages were used for the experiments. Cultures were treated daily for 3 days with either angiotensin ii alone or angiotensin ii with equimolar concentrations of saralasin. Incorporated radioactivity of [3H]thymidine and [14C]phenylalanine was measured by liquid scintillation spectrometry. RESULTS: Angiotensin ii increased [14C]phenyalanine incor-poration about 20-30%, and saralasin completely blocked the stimulation by angiotensin ii. However, there was no significant increase in [3H]thymidine incorporation by angiotensin ii stimulation in this study. CONCLUSION: These results suggest that angiotensin ii alone induces cellular hypertrophy but has no detectable mitogenic activity in cultured rat aortic VSMC.
Angiotensin II* ; Angiotensins* ; Animals ; Blood Pressure ; Collagenases ; Hypertrophy ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Pancreatic Elastase ; Radioactivity ; Rats ; Renin-Angiotensin System ; Saralasin ; Spectrum Analysis

To understand the functions of the kidney, the transcriptome of each part of the nephron needs to be profiled using a highly sensitive and unbiased tool. RNA sequencing (RNA-seq) has revolutionized transcriptomic research, enabling researchers to define transcription activity and functions of genomic elements with unprecedented sensitivity and precision. Recently, RNA-seq for polyadenylated messenger RNAs [poly(A)'-mRNAs] and classical microdissection were successfully combined to investigate the transcriptome of glomeruli and 14 different renal tubule segments. A rat kidney is perfused with and incubated in collagenase solution, and the digested kidney was manually dissected under a stereomicroscope. Individual glomeruli and renal tubule segments are identified by their anatomical and morphological characteristics and collected in phosphate-buffered saline. Poly(A)'-tailed mRNAs are released from cell lysate, captured by oligo-dT primers, and made into complementary DNAs (cDNAs) using a highly sensitive reverse transcription method. These cDNAs are sheared by sonication and prepared into adapter-ligated cDNA libraries for Illumina sequencing. Nucleotide sequences reported from the sequencing reaction are mapped to the rat reference genome for gene expression analysis. These RNA-seq transcriptomic data were highly consistent with prior knowledge of gene expression along the nephron. The gene expression data obtained in this work are available as a public Web page (https://helixweb.nih.gov/ESBL/Database/NephronRNAseq/) and can be used to explore the transcriptomic landscape of the nephron.
Animals ; Base Sequence ; Collagenases ; DNA, Complementary ; Gene Expression ; Gene Library ; Genome ; Kidney ; Microdissection ; Nephrons* ; Rats ; Reverse Transcription ; RNA* ; RNA, Messenger ; Sequence Analysis, RNA* ; Sonication ; Transcriptome*

OBJECTIVETo investigate the genotypic characterization of Porphyromonas gingivalis (Pg) and the heterogeneity of a potential virulence factor-PrtC.

METHODSArbitrarily primed polymerase chain reaction (AP-PCR) was applied to 80 Pg strains isolated from 24 unrelated Chinese periodontitis patients. PCR reaction was used to detect a fragment of the collagenase gene (PrtC gene). To evaluate the sequence heterogeneity of the Pg PrtC genes, sequence analysis of four PrtC gene of clinical isolates was performed.

RESULTSRandom primer OPA-05 and OPA-17 distinguished 7 AP-PCR profiles (I through VII). The majority of the strains belonged to type VII which accounted for 25.8%. A 548bp fragment of PrtC gene was detected from 24 clinical strains. The PCR products were verified by the restriction endonucleases PstI and PvuI. Sequence analysis showed 4 PrtC genes were heterogeneous in their nucleotide composition and differed from reference strain Pg 53977.

CONCLUSIONSThe results demonstrated genetic diversity existed among these clinical strains isolated from Chinese periodontitis patients and the PrtC genes are heterogeneous in their nucleotide sequence.

Adult ; Collagenases ; genetics ; DNA, Bacterial ; chemistry ; genetics ; Female ; Genetic Heterogeneity ; Humans ; Male ; Middle Aged ; Periodontitis ; microbiology ; Polymerase Chain Reaction ; methods ; Porphyromonas gingivalis ; genetics ; isolation & purification ; Sequence Analysis, DNA

OBJECTIVETo investigate the roles of microfilaments in the expression of collagenase and TIMP-1 in normal and hypertrophic scar.

METHODSCell culture and Northern blot hybridization methods were used in this study.

RESULTSAfter the disruption of microfilaments with cytochalasin B, mRNA contents of collagenase and TIMP-1 both increased significantly in normal and hypertrophic scar fibroblasts.

CONCLUSIONThe result suggest that the microfilament cytoskeleton may regulate the synthesis and degradation of ECM, which may be a mechanism of gene regulation during wound healing.

Actin Cytoskeleton ; physiology ; Cells, Cultured ; Collagenases ; genetics ; Fibroblasts ; metabolism ; Humans ; RNA, Messenger ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; Wound Healing ; physiology

Increased production of matrix metalloproteinases (MMPs) has been associated with increases in invasive and metastatic potential in many types of human carcinoma. Tissue inhibitors of metalloproteinase (TIMP)-1 inhibits most interstitial collagenases and MMP-9. TIMP-2 binds specifically and noncovalently to the pro-form of MMP-2 and inhibits its enzyme activity. In this study, we examined TIMP-1 and TIMP-2 expressions in relation to clinicopathological variables in colorectal carcinoma with in situ hybridization and immunohistochemistry. TIMP-1 and TIMP-2 expressions were localized overwhelmingly to pericancer stromal cells, while malignant and normal mucosal cells were weak or negative. Strong stromal TIMP-1 immunoreactivity correlated with Dukes' stage (p=0.022), status of lymph node metastasis (p=0.044) and poor survival (p= 0.005). The degree of immunohistochemical staining of TIMP-2 did not correlate with all clinicopathological variables. The correlation between enhanced TIMP-1 expression and advanced stage and poor survival suggest a growth promoting activity of TIMP-1 in colorectal carcinoma.
Adenocarcinoma/pathology ; Adenocarcinoma/mortality ; Adenocarcinoma/enzymology* ; Adult ; Aged ; Aged, 80 and over ; Antibodies ; Collagenases/immunology ; Collagenases/genetics* ; Collagenases/analysis ; Colorectal Neoplasms/pathology ; Colorectal Neoplasms/mortality ; Colorectal Neoplasms/enzymology* ; DNA Probes ; Female ; Gelatinase A ; Gelatinase B ; Gelatinases/immunology ; Gelatinases/genetics* ; Gelatinases/analysis ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Human ; In Situ Hybridization ; Male ; Metalloendopeptidases/immunology ; Metalloendopeptidases/genetics* ; Metalloendopeptidases/analysis ; Middle Age ; Predictive Value of Tests ; RNA, Messenger/analysis ; Stromal Cells/pathology ; Stromal Cells/enzymology ; Survival Analysis ; Tissue Inhibitor-of Metalloproteinase-2/immunology ; Tissue Inhibitor-of Metalloproteinase-2/genetics* ; Tissue Inhibitor-of Metalloproteinase-2/analysis ; Tissue-Inhibitor of Metalloproteinase-1/immunology ; Tissue-Inhibitor of Metalloproteinase-1/genetics* ; Tissue-Inhibitor of Metalloproteinase-1/analysis

Background and objectives: Carvedilol is a cardiovascular drug, beta- and alpha1-adrenoceptor antagonist, currently approved for the treatment of hypertension, angina, congestive heart failure by FDA. Carvedilol has been shown to attenuate oxygen free radical-initiated lipid peroxidation and to inhibit neointimal formation of aorta following vascular injury by balloon angioplasty. We have investigated the effect of carvedilol on DNA synthesis of vascular smooth muscle cells (VSMC) stimulated by platelet-derived growth factor (PDGF)-BB. MATERIALS AND METHOD: Rat aortic smooth muscle cells were obtained by the combined collagenase and elastase methods. Cells between the 4th and 8th passages were used for the experiments. Incorporated radioactivity of [3H]-thymidine was measured by liquid scintillation spectrometry. RESULTS: PDGF-BB (1 nM) increased [3H]-thymidine incorporation about 70-100% over basal value in cultured VSMC. PDGF-stimulated increase in DNA synthesis was significantly suppressed by simultaneous administration of carvedilol. In contrast, propranolol did not significantly affect 3[H]-thymidine uptake in rat aortic VSMC. CONCLUSION: The present study demonstrate that carvedilol significantly inhibits the proliferation of vascular smooth muscle cell in our condition. These results indicate that carvedilol may be effective in the treatment of cardiovascular diseases principally associated with abnormal vascular smooth muscle growth.
Angioplasty, Balloon ; Animals ; Aorta ; Cardiovascular Diseases ; Cell Proliferation ; Collagenases ; DNA ; Heart Failure ; Hypertension ; Lipid Peroxidation ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Oxygen ; Pancreatic Elastase ; Platelet-Derived Growth Factor ; Propranolol ; Radioactivity ; Rats ; Spectrum Analysis ; Vascular System Injuries

PURPOSE: Transient hypoxia is an initial event that accentuates ischemia/reperfusion (I/R) injury in the liver. Hepatic ischemia/reperfusion (I/R) injury is largely related to innate immunity via Toll-like receptor 4 (TLR4) signaling. However, the mechanism by which hypoxia could lead to activate TLR4 signaling remains unclear. Therefore, the aim of this experimental study investigates how TLR4 signalling is activated by hypoxia. METHODS: Hepatocytes were isolated from male wild-type (C57BL/6) mice (8~12 weeks old) by an in situ collagenase (Type IV, Sigma-Aldrich) perfusion technique. In this study, using primary mouse hepatocytes in culture to 1% oxygen, detection of TLR4 translocation to the lipid rafts on the cell membrane by immunofluorescence staining and immunoblotting was saught. RESULTS: Hypoxia caused TLR4/MD2 and beta2-Integrin (CD11b/CD18) translocation to lipid rafts associated with CD14 in hepatocytes. The cholesterol sequestering agent, Nystatin and Filipin prevented hypoxia-induced TLR4/MD2 translocation to lipid rafts. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of hepatocytes similarly caused TLR4/MD2 translocation to lipid rafts. In addition, translocation of hypoxia-induced TLR4 complex was inhibited by N-acetylcysteine (NAC) demonstrating that the activation of TLR4 signaling is dependent on ROS. Further, the cholesterol sequestering agent, nystatin, prevented hypoxia-induced high mobility group box 1 (HMGB1) release in hepatocytes. CONCLUSION: These results suggest that ROS dependent TLR4 signaling is achieved following receptor translocation to the lipid raft in hepatocytes. We hypothesized that this mechanism is required for the release of HMGB1, an early mediator of injury and inflammation in hepatic I/R injury.
Acetylcysteine ; Animals ; Anoxia* ; Cell Membrane ; Cholesterol ; Cluster Analysis* ; Collagenases ; Filipin ; Fluorescent Antibody Technique ; Hepatocytes* ; HMGB1 Protein ; Humans ; Immunity, Innate ; Immunoblotting ; Inflammation ; Liver ; Male ; Mice* ; Nystatin ; Oxidative Stress ; Oxygen ; Perfusion ; Sequestering Agents ; Toll-Like Receptor 4*

BACKGROUNDNo efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.

METHODSReal-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type I collagen. In addition to hepatic hydroxyproline content, hepatic collagen types I and III were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.

RESULTSExogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P < 0.01), hepatic hydroxyproline content and the accumulation of collagen types I and III were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P < 0.01) as compared with the model group.

CONCLUSIONThe results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.

Animals ; Antisense Elements (Genetics) ; therapeutic use ; Collagenases ; metabolism ; Hydroxyproline ; analysis ; Liver ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; therapy ; Male ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; antagonists & inhibitors ; genetics

Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin-dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolytic activity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability.
Chlorhexidine ; chemistry ; pharmacology ; Collagenases ; pharmacology ; Dental Bonding ; Dental Cements ; chemistry ; Dental Stress Analysis ; instrumentation ; Dentin ; drug effects ; ultrastructure ; Dentin-Bonding Agents ; chemistry ; Gelatinases ; pharmacology ; Humans ; Hydroxyproline ; analysis ; Matrix Metalloproteinase 8 ; pharmacology ; Matrix Metalloproteinase Inhibitors ; chemistry ; pharmacology ; Proanthocyanidins ; chemistry ; pharmacology ; Stress, Mechanical ; Surface Properties ; Tensile Strength ; Tooth Demineralization ; pathology ; physiopathology