Objective To explore the effect of overwork (OW) on extracellular matrix of arterial vessel wall in rats. Methods Random number grouping method was employed to assign 18 Sprague-Dawley rats into three groups(n=6):the control group(no special treatment),group OW(forced swimming twice a day for 15 days),and sleep deficiency(SD)+OW group(in addition to forced swimming twice a day,the rats were put on the platforms in water to limit sleep for 15 days).On the 16th day,the abdominal aorta and common carotid artery were collected after blood sampling from heart under deep anesthesia.A part of the abdominal aorta sample was taken for Masson staining of collagen fiber,and Verhoeff-Van Gieson staining was carried out for the elastic fiber of common carotid artery.Image J was employed for the quantitative analysis of collagen fiber and elastic fiber content.The expression of collagen 1(Col-1) protein was quantified by immunohistochemistry and the ultrastructure of vascular matrix was examined by transmission electron microscopy.The other part of the abdominal aorta sample was used to determine the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,tissue inhibitor of metalloproteinases-1(TIMP-1),and Col-1 by quantitative real-time polymerase chain reaction. Results Compared with that in control group,the content of collagen fiber in groups OW and SD+OW had no significant change(all P>0.05);the content of elastic fiber in groups OW and SD+OW decreased(all P<0.001) and had no significant difference between each other(P>0.05).The vascular vessel wall of group OW showed slight fiber breakage,while that of group SD+OW presented wormhole-like or spongy fiber fragmentation.The mRNA levels of MMP-1 and MMP-2 in groups OW and SD+OW had no significant difference between each other(P>0.05) but were higher than that in control group(all P<0.001).The mRNA levels of MMP-9 and TIMP-1 had no significant difference among the three groups(all P>0.05).Groups OW and SD+OW had lower mRNA level(all P<0.001) and protein level(all P<0.001) of Col-1 than control group,while the mRNA and protein levels of Col-1 had no significant difference between groups OW and SD+OW(P>0.05). Conclusion OW can reduce the content of Col-1 and elastic fibers in the extracellular matrix of arterial vessels,destroy the elastic lamina of vascular wall,up-regulate the expression of MMP-1 and MMP-2,thereby injuring arterial vessels.
Animals ; Collagen Type I ; Extracellular Matrix/metabolism* ; Matrix Metalloproteinase 1/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; RNA, Messenger/genetics* ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1/metabolism*

BACKGROUNDExposure of adult mice to more than 95% O2 produces a lethal injury by 72 hours. Nuclear factor kappa B (NF-κB) is a transcriptional factor that plays a key role in the modulation of cytokine networks during hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. Studies have reported that exogenous OPN can maintain the integrity of the cerebral microvascular basement membrane and reduce brain damage through inhibiting NF-κB activities in the brain after subarachnoid hemorrhage. However, it is not clear whether OPN can reduce lung injury during ALI by inhibiting transcriptional signal pathways of NF-κB and consequent inhibition of inflammatory cytokines. Thus we examined the effects and mechanisms of recombinant OPN (r-OPN) on ALI.

METHODSNinety-six mice were randomly divided into phosphate buffered saline (PBS) and r-OPN groups. Mice were put in an oxygen chamber (>95% O2) and assessed for lung injury at 24, 48, and 72 hours. Expressions of NF-κB, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), and tissue inhibitors of MMP-2 and MMP-9 (TIMP-1, TIMP-2) mRNA in lungs were examined with RT-PCR. Expression and distribution of NF-κB protein in lungs were measured with immunohistochemistry.

RESULTSExposure to hyperoxia for 72 hours induced more severe lung injury in the PBS group compared with the r-OPN group. Expression of NF-κB mRNA in the PBS group exposed to hyperoxia for 48 and 72 hours was significantly higher than the r-OPN group (P < 0.05). With 72-hour exposure, expression of TIMP-1 mRNA in the r-OPN group was significantly higher than that of the PBS group (P < 0.05). Expression of TIMP-2 mRNA in the r-OPN group at 48 and 72 hours was significantly higher than those in the PBS group (P < 0.05). After 72-hour exposure, expression of NF-κB protein in airway epithelium in the PBS group was significantly higher than that in the r-OPN group (P < 0.05).

CONCLUSIONr-OPN can inhibit the release and activation of MMPs through inhibition of the expression of NF-κB and promotion of the expression of TIMPs, and alleviate hyperoxia-induced ALI.

Acute Lung Injury ; genetics ; metabolism ; Animals ; Hyperoxia ; metabolism ; physiopathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice ; NF-kappa B ; genetics ; metabolism ; Osteopontin ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism

Objective: To explore the effect of microRNA(miR)-222 on cell proliferation and apoptosis of fibroblasts in hypertrophic scar (HS) and the underlying mechanisms. Methods: The expression of miR-222 in the HS and the normal skin tissues was detected by real-time RT-PCR. The HS fibroblasts were transfected with miR-222 mimic and miR-222 inhibitor respectively. The cell viability was tested with MTT assay, cell cycle distribution and apoptosis were detected with flow cytometry and the expression levels of proliferation, apoptosis and cell cycle related proteins were determined with Western blot. Direct target of miR-222 was evaluated by dual-luciferase reporter assay. Results: miR-222 was significantly up-regulated in HS tissues compared with normal skin tissues(P<0.05). Overexpression of miR-222 enhanced the cell viability of HS fibroblasts; increased mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), collagen alpha-1 (Ⅰ) chain (Col1A1) and collagen alpha-1 (Ⅲ) chain (Col3A1); increased cell population in S phase and protein expressions of cyclin D1, cyclin E1 and cyclin-dependent kinases 1 (CDK1); inhibited cell apoptosis and reduced protein expressions of caspase-3/9. Overexpression of MMP1 attenuated the effects of miR-222 on the cell viability and apoptosis in fibroblasts, reduced expression levels of PCNA, cyclin D1 and the expression of caspase-3 was increased. Conclusion: miR-222 enhances cell proliferation and inhibits cell apoptosis of HS fibroblasts through negative regulation of MMP1, which suggests that miR-222 and MMP1 might be used as novel biomarkers and targets in diagnostic and therapeutic approaches for HS.
Apoptosis ; genetics ; Cell Proliferation ; genetics ; Cicatrix, Hypertrophic ; Fibroblasts ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; MicroRNAs ; metabolism

OBJECTIVETo study the association between the functional polymorphism of matrix metalloproteinases (MMPs) and the development of chronic obstructive pulmonary disease (COPD).

METHODS147 COPD patients and 120 healthy smoking controls were selected. Spirometry and chest X-rays had been taken. Questionnaires including sex, age, smoking history, occupational exposure were completed. MMP-9 (-1562 C/T), MMP-1(-1607 1G/2G), MMP-12 (-82 A/G), MMP-12(-357 Asn/ Ser) alleles were determined using PCR-RFLP method. Independent samples T test analysis was carried out to compare patients' age, smoking index, FEV1 /FVC, FEV1 % pred with that of healthy controlled group. The frequencies of genotypes and alleles between groups were analyzed by chi-square tests and multilogistic regression.

RESULTSMMP12 Asn/Asn, CT/AsnAsn were risk factors for smoking-induced COPD. The ORs were 2.361 (95% CI: 1.369-4.017) and 2.433(95% CI: 1.159-5.342) respectively while CC/1G1G/ SerSer seemed to be a protective factor for smoking-induced COPD, with OR as 0.457 and 95% CI as 0.231-0.911.

CONCLUSIONAsn/Asn, CT/AsnAsn might be susceptible genotypes while CC/GG/SerSer might serve as protective genotype.

Aged ; Case-Control Studies ; China ; ethnology ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Logistic Models ; Male ; Matrix Metalloproteinase 1 ; genetics ; Matrix Metalloproteinase 12 ; genetics ; Matrix Metalloproteinase 9 ; genetics ; Polymorphism, Genetic ; Pulmonary Disease, Chronic Obstructive ; genetics

OBJECTIVETo investigate the mechanism of matrix metalloproteinases (MMP) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes.

METHODSPg ATCC 33277 (type I), WCSP115 (type II), WCSP1.5 (type III), W83 (type IV) were assessed for their inductions of MMP-1 and MMP-2 expression in HGF. MMP mRNA levels of HGF were determined by real-time RT-PCR and MMP protein levels in culture supernatant were determined by ELISA at different time intervals (1, 3, 6 and 12 h) following continuous co-culture of bacteria with HGF.

RESULTSWhen co-cultured with Pg, the MMP-1 and MMP-2 mRNA and protein expression of HGF significantly increased compared with the negative control group (P < 0.01). The group of type II showed greater up-regulated than other fimA genotypes in the mRNA and protein expressions of MMP-1 and MMP-2, MMP-1 mRNA [(28.88 +/- 3.12) - (231.01 +/- 24.99)] and protein [(1.35 +/- 0.17) - (3.08 +/- 1.20)] microg/L; MMP-2 mRNA [(20.42 +/- 2.21) - (188.34 +/- 37.37)] and protein [(2.57 +/- 0.76) - (18.08 +/- 1.15)] microg/L for different time periods; While the group of type III was weaker than other fimA genotypes, the level of MMP-1 mRNA was [(5.11 +/- 0.55) - (72.84 +/- 8.84)] and protein [(0.68 +/- 0.13) - (1.46 +/- 0.94)] microg/L, MMP-2 mRNA [(4.55 +/- 0.55) - (25.75 +/- 3.12)] and protein [(2.28 +/- 0.93) - (11.22 +/- 2.46)] microg/L (P < 0.05).

CONCLUSIONSPg could induce HGF to over-express MMP, and fimA genotypes of Pg may be related to this pathogenicity, which might indicate fimA genotype is associated with pathogenesis of Pg.

Cells, Cultured ; Coculture Techniques ; Fibroblasts ; metabolism ; Fimbriae Proteins ; genetics ; Genotype ; Gingiva ; cytology ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Porphyromonas gingivalis ; genetics ; RNA, Messenger ; genetics

OBJECTIVETo study the role of the synthesis and degradation of collagen at the transcription level during liver fibrogenesis due to schistosomiasis japonica in rabbits.

METHODSNew Zealand rabbits challenged by cercariae of Schistosoma japonicum (S. japonicum) were served as animal models for liver fibrosis. Liver specimens were collected through operations at 4, 6, 8, 10, 12, 16, 20, 24 and 28 wks after challenge. Type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels of liver tissue were detected by RT-PCR + Dot blot. The size of egg granulomas and the degree of liver fibrosis were measured by histopathological examinations.

RESULTSType I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels increased simultaneously in the early stage after challenge. Most of them reached their peak at 10 weeks, and compared with normal controls, type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels increased by 12.0-, 11.0-, 6.6-, 10.0- and 11.0-fold, respectively, coinciding with the change of egg granulomas, i.e., the change in the inflammatory process. Then both collagen and collagenase mRNA levels decreased. Type I, III and IV collagen mRNA levels declined to 2-fold to 3-fold as compared with normal controls (P < 0.05), while MMP-1 and MMP-9 mRNA levels declined close to normal levels (P > 0.05) at 28 wks. This study shows that the synthesis and degradation of collagen keep a dynamic balance at the early stage of schistosomiasis japonica challenge, while at the later stages the quantity of collagen synthesis was higher than that of collagen degradation.

CONCLUSIONSIt was confirmed at transcription level that when the quantity of collagen synthesis was higher than that of collagen degradation liver fibrogenesis may be resulted in.

Animals ; Collagen ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; Matrix Metalloproteinase 1 ; genetics ; Matrix Metalloproteinase 9 ; genetics ; RNA, Messenger ; analysis ; Rabbits ; Schistosomiasis japonica ; metabolism ; Transcription, Genetic

OBJECTIVETo observe the effects of Dahuang Zhechong Pill (DZP) on the gene expression of matrix metalloproteinase-1 (MMP-1) in rats' hepatic stellate cells (HSC) and activity of matrix metalloproteinase-2 (MMP-2) that secreted into the culture base.

METHODSHSC were isolated from the liver of normal rats and incubated with DZP-contained drug serum. The expression of MMP-1 in the HSC was detected by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The activity of MMP-2 was examined by zymography.

RESULTSDZP-contained drug serum could obviously promote the gene expression of MMP-1 in HSC. In the meantime, it could obviously increase the content and activity of MMP-2 synthesized by HSC (P < 0.05).

CONCLUSIONThe anti-fibrosis action of DZP was correlated to the promotion of HSC's gene expression of MMP-1 and increasing of the contents and activity of MMP-2.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Hepatocytes ; cytology ; metabolism ; Male ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

We investigated the expression of membrane type-1 (MT1)-MMP, MMP2, MMP9 and TIMP2 mRNAs and their roles in ductal carcinoma in situ (DCIS) and T1 and T2 invasive ductal carcinoma of the breast. We further compared these two types of carcinomas for differences in microvessel density, and expression of angiogenic factors and CD44std. MT1-MMP, MMP2, MMP9 and TIMP2 mRNA were expressed in both DCIS and invasive ductal carcinomas. Expression rates of MT1-MMP, MMP2, MMP9 and TIMP2 mRNAs were not statistically different between DCIS and invasive ductal carcinomas, nor did they differ statistically when grouped by tumor size, histologic grade or nuclear grade of invasive ductal carcinoma. Microvessel density and expression of VEGF and TGF-beta were not statistically different between DCIS and invasive ductal carcinoma. CD44std expression was significantly increased in DCIS compared to invasive ductal carcinoma (p < 0.05) and it was also significantly increased in lower clinical stage, histologic grade and nuclear grade of invasive ductal carcinoma (p < 0.05). Axillary node metastasis was significantly correlated with MT1-MMP mRNA, VEGF and TGF-beta expression (p < 0.05) and MT1-MMP mRNA was positively correlated with VEGF expression and TIMP2 mRNA (p < 0.05). In summary, patterns of MMP mRNA expression in DCIS and invasive ductal carcinoma suggest that the invasive potential of breast carcinoma is already achieved before morphologically overt invasive growth is observed. As MT1-MMP mRNA expression is significantly correlated with axillary nodal metastasis, it may be useful as a prognostic indicator of invasive ductal carcinoma. Considering the positive correlation of MT1-MMP mRNA and TIMP2mRNA expression, our finding supports a role for TIMP2 in tumor growth, as well as the utility of CD44std as a prognostic indicator of breast cancer.
Tissue Inhibitor of Metalloproteinase-2/genetics ; RNA, Messenger/metabolism ; Matrix Metalloproteinases, Membrane-Associated ; Matrix Metalloproteinases/*genetics ; Matrix Metalloproteinase 9/genetics ; Matrix Metalloproteinase 2/genetics ; Matrix Metalloproteinase 1/genetics ; Humans ; Gene Expression Regulation, Neoplastic ; Gene Expression Regulation, Enzymologic ; Female ; Carcinoma, Ductal, Breast/genetics/*physiopathology ; Carcinoma in Situ/genetics/*physiopathology ; Breast Neoplasms/genetics/*physiopathology

We investigated the expression of membrane type-1 (MT1)-MMP, MMP2, MMP9 and TIMP2 mRNAs and their roles in ductal carcinoma in situ (DCIS) and T1 and T2 invasive ductal carcinoma of the breast. We further compared these two types of carcinomas for differences in microvessel density, and expression of angiogenic factors and CD44std. MT1-MMP, MMP2, MMP9 and TIMP2 mRNA were expressed in both DCIS and invasive ductal carcinomas. Expression rates of MT1-MMP, MMP2, MMP9 and TIMP2 mRNAs were not statistically different between DCIS and invasive ductal carcinomas, nor did they differ statistically when grouped by tumor size, histologic grade or nuclear grade of invasive ductal carcinoma. Microvessel density and expression of VEGF and TGF-beta were not statistically different between DCIS and invasive ductal carcinoma. CD44std expression was significantly increased in DCIS compared to invasive ductal carcinoma (p < 0.05) and it was also significantly increased in lower clinical stage, histologic grade and nuclear grade of invasive ductal carcinoma (p < 0.05). Axillary node metastasis was significantly correlated with MT1-MMP mRNA, VEGF and TGF-beta expression (p < 0.05) and MT1-MMP mRNA was positively correlated with VEGF expression and TIMP2 mRNA (p < 0.05). In summary, patterns of MMP mRNA expression in DCIS and invasive ductal carcinoma suggest that the invasive potential of breast carcinoma is already achieved before morphologically overt invasive growth is observed. As MT1-MMP mRNA expression is significantly correlated with axillary nodal metastasis, it may be useful as a prognostic indicator of invasive ductal carcinoma. Considering the positive correlation of MT1-MMP mRNA and TIMP2mRNA expression, our finding supports a role for TIMP2 in tumor growth, as well as the utility of CD44std as a prognostic indicator of breast cancer.
Tissue Inhibitor of Metalloproteinase-2/genetics ; RNA, Messenger/metabolism ; Matrix Metalloproteinases, Membrane-Associated ; Matrix Metalloproteinases/*genetics ; Matrix Metalloproteinase 9/genetics ; Matrix Metalloproteinase 2/genetics ; Matrix Metalloproteinase 1/genetics ; Humans ; Gene Expression Regulation, Neoplastic ; Gene Expression Regulation, Enzymologic ; Female ; Carcinoma, Ductal, Breast/genetics/*physiopathology ; Carcinoma in Situ/genetics/*physiopathology ; Breast Neoplasms/genetics/*physiopathology

OBJECTIVEWe investigated the in vivo effects of recombinant adenovirus-associated virus type-2 (AAV-2) mediated interleukin-10 (IL-10) gene transfer on the expression of matrix metalloproteinase (MMP)-2, 9, tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I and type III in a rat acute myocardial infarction model.

METHODMale Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 6): sham operation group, MI/AAV2 group, and MI/AAV2-IL-10 group (10(10) vg/ml x 0.1 ml injection at peri-infarct regions immediately post MI). Five days later, the expressions of MMP-2 and MMP-9 were measured by RT-PCR, Western blot and zymography. The expression of TIMP-1 was measured by RT-PCR and Western blot. Collagen type I and type III were assessed by RT-PCR and immunohistochemical stain.

RESULTSThe myocardial expressions of MMP-2, MMP-9 and collagen contents in MI/AAV2 group were significantly increased than those in sham operation group. Myocardial expressions of MMP-2, MMP-9 were significantly decreased and the expression of TIMP-1 significantly increased in the MI/AAV2-IL-10 group than those in MI/AAV2 group. Moreover, the expressions of collagen type I, collagen type III and the ratio of I/III collagen in border zones of infarcted myocardium were decreased by 47.6% (P < 0.01), 23.6% (P < 0.05), and 17.9% (P < 0.05) respectively, while the expression of TIMP-1 increased by 73.1%(P < 0.05) in MI/AAV2-IL-10 group compared to MI/AAV2 group.

CONCLUSIONIn vivo myocardial IL-10 transfer reduced myocardial MMP and collagen expression and increasing the TIMP expression.

Animals ; Extracellular Matrix ; metabolism ; Gene Expression ; Genetic Therapy ; Interleukin-10 ; genetics ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Myocardial Infarction ; genetics ; metabolism ; physiopathology ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transfection ; Ventricular Remodeling