mRNA Levels Of Metalloproteinases Research Articles

Evaluation of Virola oleifera activity in musculoskeletal pathologies: Inhibition of human multiple myeloma cells proliferation and combination therapy with dexamethasone or bortezomib

Ethnopharmacological relevanceVirola oleifera (Schott) A.C. Smith, Myristicaceae, has been widely used in traditional medicine in Brazil to treat rheumatic pain, joint tumours, skin diseases, halitosis, bronchial asthma, haemorrhoids, and intestinal worms. Recently, research data showed the antioxidant properties in several oxidative stress-related models. However, there is no experimental evidence supporting its potential use in managing rheumatic diseases and bone malignancies. Aims of the studyTo evaluate the therapeutic potential of the resin from Virola oleifera in joint and bone diseases, namely arthritis, osteosarcoma, chondrosarcoma, and multiple myeloma. Materials and methodsTo determine Virola oleifera resin (VO) effects on arthritis-associated inflammation and cartilage degradation, the LPS-induced NO production, and mRNA and protein expression of ADAMTS5, MMP13, COL2, and ACAN, were evaluated in chondrocytes (ATDC5 and TC28 cell lines). The cytotoxic effects of VO (0.05–50 μg/ml) on multiple myeloma (ARH-77), osteosarcoma (SAOS-2), and chondrosarcoma (SW-1353) cell lines were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The VO effects, combined with dexamethasone or bortezomib, were evaluated in a multiple myeloma cell line. The mechanisms of VO, alone or in combination with bortezomib, were determined by cell cycle analysis through flow cytometry, while expression levels of p-Akt/Akt, p-ERK/ERK, p-p38/p38 MAPK, Bax, Bcl-2, and cleaved-caspase-3/caspase-3 proteins by Western blot. ResultsVO had no significant effect on LPS-induced NO production in chondrocytes at non-cytotoxic concentrations. VO treatment diminished the mRNA levels of metalloproteinases and ECM components; however, any significant effect was observed on the protein expression levels. The cell viability of a multiple myeloma cell line was strongly reduced by VO treatment in a dose- and time-dependent manner, while osteosarcoma and chondrosarcoma cell lines viability was significantly affected only by the highest dose assessed. In multiple myeloma cells, VO leads to G2/M cell cycle arrest. Furthermore, it synergizes with dexamethasone by increasing cell toxicity. Finally, VO reverts bortezomib activity by counteracting ERK1/2, Bax, and caspase-3 activation. ConclusionsThe current work supports the ethnopharmacological use of Virola oleifera (Schott) A.C. Smith in bone and joint diseases, but there is no evidence for the amelioration of arthritis-associated inflammatory or catabolic processes. Our data also supports the potential use of Virola oleifera as adjuvant therapy to optimize the pharmacologic effects of current chemotherapeutic drugs. However, possible herb-drug interactions should be considered before clinical application.

P863Morphometric analysis of the dynamic changes of the interstitium after reperfused myocardial infarction

Abstract Background The interstitial space is mainly composed by cells, fibers and gels of polysaccharides, which act as a compression buffer against the stress placed on the extracellular matrix (ECM). After myocardial infarction (MI), heart has to withstand higher mechanical stress due to injured cardiomyocytes. ECM composition notably influences the mechanical properties of the myocardium and participates in left ventricular remodeling. Purpose To characterize the myocardial ECM changes from ischemia onset until late phases after coronary reperfusion in a swine model of reperfused MI. Methods MI was induced in swine by transient 90-min coronary occlusion using angioplasty balloons. One control group and three MI groups were defined: 1) without reperfusion, 2) 1-week, and 3) 1-month reperfusion (n=5, each). Myocardial samples from the infarcted area were isolated and histologically staining to evaluate the presence of collagen type I, collagen type III, elastic fibers, and proteinglycans. Moreover, the presence of laminin, fibronectin, and secreted protein, acidic and rich in cysteine (SPARC) was determined by immunohistochemistry. Five independent photographs for each group and each stain were taken and the presence of the different components of the interstitium was morphometrically quantified. Indeed, the mRNA levels of metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), and transcription factors implicated in ECM remodeling were quantified. Results In the no-reperfusion group, an increase in fibronectin, laminin, and elastic fibers were observed, whereas no changes neither in the quantity nor in the organization of collagen-I and collagen-III fibers were detected. In the 1-week and 1-month reperfusion groups, an augmentation in the content of collagen-I, collagen-III, elastic fibers were found. These fibers displayed a more organized pattern compared to control tissue. The quantity of proteinglycans, laminin, fibronectin, and SPARC were also increased. These changes could be explained by a heightening in the mRNA expression of TIMP1, TIMP2, and TIMP3 from the ischemic period until chronic phases, whereas no changes were observed for TIMP4. MMP2 and MMP9 mRNA levels were boosted in late phases post-MI. When exploring the mRNA levels of transcription factors implicated in ECM remodeling, CTGF increased during the ischemic period, whereas TNF-α and TGF-β augmented after 1-week and 1-month post-reperfusion, respectively. Conclusions ECM remodeling starts after ischemia onset, probably aiming to protect cardiomyocytes from the ischemic damage. After reperfusion, the ECM evolves to constitute a fibrotic scar to maintain a proper cardiac function. These changes may be orchestrated by the fluctuation in genes specially implicated in ECM distribution. ECM changes might be accurately regulated since it could provoke an adverse left ventricular remodelling, consequently impairing patient prognosis. Acknowledgement/Funding This study was funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (PIE15/00013, PI17/01836, and CIBERCV16/11

Effects of polycystin‑1 N‑terminal fragment fusion protein on the proliferation and apoptosis of rat mesangial cells.

Mesangial proliferative glomerulonephritis (MsPGN) is characterized by widespread mesangial cell proliferation and an accumulation of extracellular matrix (ECM) in the mesangial area. In a previous study we developed a polycystin‑1 N‑terminal fragment (PC‑1 NF) fusion protein that inhibits the proliferation of cyst‑lining epithelial cells in autosomal dominant polycystic kidney disease. In addition, the PC‑1 NF fusion protein arrests the cell cycle of cancer cells at the G0/G1 phase, inhibiting their proliferation. In the present study, the effect of the PC‑1 NF fusion protein on MsPGN was investigated. It was found that the PC‑1 NF fusion protein inhibited the proliferation of rat mesangial cells and induced G0/G1 phase arrest and apoptosis invitro. PC‑1 NF fusion protein treatment also resulted in a decrease in mRNA expression levels of proliferating cell nuclear antigen, cyclinD1 and B‑cell lymphoma‑2 (Bcl‑2) and an increase in mRNA expression levels of Bcl‑2‑associated X protein (Bax) and p21Waf1. Furthermore, a decrease in Bcl‑2, c‑fos, c‑jun and protein kinase C‑α protein levels was observed, whereas Bax protein levels increased. Additionally, PC‑1 NF fusion protein induced ECM degradation and inhibited ECM expansion. The results also demonstrated that PC‑1 NF fusion protein treatment resulted in a decrease in typeIV collagen and tissue inhibitor of metalloproteinase mRNA levels but an increase in matrix metalloproteinase2 mRNA levels. In combination, these results suggest that the PC‑1 NF fusion protein inhibits proliferation, promotes apoptosis and induces ECM degradation in MsPGN rats. This study offers novel perspectives for the treatment of MsPGN.

Recombinant Gelatin Microspheres: Novel Formulations for Tissue Repair?

Microspheres (MSs) can function as multifunctional scaffolds in different approaches of tissue repair (TR), as a filler, a slow-release depot for growth factors, or a delivery vehicle for cells. Natural cell adhesion-supporting extracellular matrix components like gelatin are good materials for these purposes. Recombinant production of gelatin allows for on-demand design of gelatins, which is why we aim at developing recombinant gelatin (RG) MSs for TR. Two types of MSs (50 < Ø < 100 microm) were prepared by crosslinking two RGs, Syn-RG, and the arginine-glycine-aspartate-containing Hu-RG. The MSs were characterized, and their tissue reaction and degradation in rats was examined. Histological analysis of the explants after 14 and 28 days in vivo also showed that Syn-RG was degraded slower than Hu-RG, which correlated with the in vitro degradation assay. Hu-RG explants displayed more cellular ingrowth (60% vs. 15% for Syn-RG at day 14), which was associated with extracellular matrix deposition and vascularization. The infiltrating cells consisted of mainly macrophages, part of which fused to giant cells locally, and fibroblasts. No differences were found in matrix metalloproteinase mRNA levels, whereas gelatinase activity was clearly higher in Hu-RG explants. In conclusion, the in vitro and in vivo results of these novel formulations pave the way for cell- and/or factor-driven TR by these RG MSs.

Matrix Metalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase mRNA Levels are Specifically Altered in Torn Rotator Cuff Tendons

Background Rotator cuff tears are a cause of pain and disability in the shoulder. The molecular changes associated with rotator cuff tearing are unclear. A subset of matrix metalloproteinases and tissue inhibitors of metalloproteinase, which are involved in extracellular matrix remodeling and degradation, were evaluated. Hypothesis There would be an increase in the mRNA level of spcific matrix metalloproteinase and a decrease in the mRNA level of specific tissue inhibitors of metalloproteinase in rotator cuff tendon tissue obtained from patients with rotator cuff tears. Study Design Controlled laboratory study. Methods Tissue was obtained from 10 patients undergoing rotator cuff repair for full-thickness rotator cuff tears. Also, tissue was obtained from cadaveric specimens with no gross evidence of rotator cuff tearing. Reverse transcription polymerase chain reaction was performed for the collagenases (MMP-1, MMP-8, MMP-13), the stromelysins (MMP-3, MMP-10, MMP-11), and the tissue inhibitors of metalloproteinase (TIMP-1, TIMP-2, TIMP-3, TIMP-4). Western blotting was performed to confirm the mRNA changes demonstrated in collagenase-3 (MMP-13). Results There was a significant increase in collagenase-3 (MMP-13) mRNA levels, a decrease in stromelysin-1 (MMP-3) mRNA levels, and a decrease in tisue inhibitor of metalloproteinase-2, -3, and -4 mRNA levels. Western blotting demonstrated an increase in the active form of collagenase-3 (MMP-13) in rotator cuff tendon tears. Conclusions The mRNA levels of specific matrix metalloproteinases and tissue inhibitors of metalloproteinase are altered in torn rotator cuff tendons. Clinical Relevance With the known action of the matrix metalloproteinases and tissue inhibitors of metalloproteinase in extra-cellular matrix remodeling, these findings suggest that their roles in remodeling of rotator cuff tears should be further investigated.

Recombinant connective tissue growth factor modulates porcine skin fibroblast gene expression.

Connective tissue growth factor (CTGF) is a 38 Kda cysteine-rich, heparin-binding peptide that has been implicated in several normal and abnormal physiological processes. CTGF has been shown to be induced by transforming growth factor-beta. Previous studies in our pig model of skin wound healing showed a coordinate expression of transforming growth factor-beta and CTGF during the healing process. To better understand the function of CTGF during wound healing, normal porcine fibroblasts were isolated from skin samples from SPF Yorkshire pigs. At fourth passage the cells were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum and at 80% confluence the medium was replaced with supplemented serum-free medium. After a further 24 hours, cells were treated with 0, 10, 25, 50, 100, and 500 ng/ml of 38 Kda or 16-20 Kda (C-terminal truncated form) recombinant expressed human CTGF for 24 hours or treated with 100 ng/ml for 0, 12, 24, and 48 hours. Subsequently, CTGF effects on cell DNA synthesis and mRNA levels for a subset of relevant molecules were assessed. The results showed that in cells treated with 38 Kda rhCTGF, mRNA levels for types I and III collagen, fibromodulin, and basic fibroblast growth factor were significantly up-regulated, but mRNA levels for HSP47, decorin, biglycan, and versican were not significantly altered. mRNA levels for CTGF were also significantly increased, indicating autoregulation of expression. However, mRNA levels for transforming growth factor-beta, inteleukins 1 and 6, tumor necrosis factor-alpha, and nerve growth factor did not change. Interestingly, mRNA levels for the tissue inhibitors of metalloproteinase-1, -2, -3 and -4 were observed to significantly increase, but in contrast, mRNA levels for matrix metalloproteinases-1, -2, -9 were not significantly altered by exposure of the cells to the 38 Kda form of CTGF. In addition, DNA synthesis was augmented in the presence of 38 Kda rhCTGF. However, the truncated 16-20 Kda form of rhCTGF appeared to have none of these effects on porcine fibroblasts. These results indicate that in order to induce changes in porcine fibroblasts a molecule with an intact C-terminal domain is required, and that CTGF regulates porcine fibroblast extracellular matrix molecule, growth factor, and proteinase inhibitor gene expression without apparently affecting matrix metalloproteinase mRNA levels. These findings suggest that CTGF contributes to the anabolic environment during skin wound healing via selective modulation of fibroblast proliferation and changes to gene expression.

Angiotensin II type 1 and endothelin type A receptor antagonists modulate the extracellular matrix regulatory system differently in diastolic heart failure.

Ventricular fibrosis plays a pivotal role in the development of diastolic heart failure and is a therapeutic target; however, the effects of pharmacological interventions on the extracellular matrix (ECM) regulatory system in diastolic heart failure remain to be clarified. Dahl salt-sensitive rats fed on a diet containing 8% NaCl from age 7 weeks--a hypertensive diastolic heart failure model--were divided into untreated rats, rats treated with angiotensin II type 1 (AT1) receptor antagonist and those treated with endothelin type A (ETA) receptor antagonist. Ventricular fibrosis progressed in the untreated rats, with increases in mRNA levels of type I collagen, matrix metalloproteinase-2, -9 and -13, and tissue inhibitor of metalloproteinase-1 and -2. Both antagonists attenuated ventricular fibrosis to the same degree. AT1 receptor blockade decreased the type I collagen mRNA level more than ETA receptor blockade. ETA receptor blockade did not decrease the matrix metalloproteinase-2 mRNA level that was decreased by AT1 receptor blockade, and decreased the tissue inhibitor of metalloproteinase-2 mRNA level that was not affected by AT1 receptor blockade. These led to a higher ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 mRNA levels and a greater 72-kDa gelatinase activity in the rats treated with ETA receptor antagonist than in those treated with AT1 receptor antagonist, and may well cancel out the lesser decline in collagen synthesis, resulting in the equivalent attenuation of ventricular fibrosis. AT1 receptor and ETA receptor antagonists provide their beneficial effects on the ECM through different modulation of its regulatory system.

Furin and membrane type-1 metalloproteinase mRNA levels and activation of metalloproteinase-2 are associated with arterial remodeling

Matrix metalloproteinase (MMP) activation is an essential feature of pathological and physiological arterial enlargement or shrinkage. Recently, furin-activated membrane type-1 MMP (MT1-MMP) was identified as the in vivo activator of MMP2 in mice. Although arterial enlargement and shrinkage are important in several pathological processes, this proprotein convertase–MT1-MMP axis has not been described during arterial remodeling. In rabbit femoral and carotid arteries, we report an increase in furin and MT1-MMP mRNA levels before and at the onset of arterial remodeling followed by an increase in activated MMP2. This reveals the presence of the proprotein convertase–MT1-MMP axis in flow-induced arterial remodeling and identifies furin as a possible target for local intervention in pathological arterial remodeling.

Wild-Type p53 Gene Transfer Inhibits Invasion and Reduces Matrix Metalloproteinase-2 Levels in p53-Mutated Human Melanoma Cells

The tumor suppressor gene p53 has inhibitory effects on cell growth and angiogenesis and induces apoptosis when overexpressed in melanoma and in a variety of tumor cells by adenovirus-mediated gene transfer. The invasive ability of tumor cells, facilitating local infiltration and metastasis, is related to matrix metalloproteinase levels. In melanoma, matrix metalloproteinase-2 and matrix metalloproteinase-9 have a prominent role in this process. The aim of this study was to evaluate whether wild-type p53 overexpression, obtained by a recombinant adenovirus vector (AdCMV.p53), affects cell invasiveness through modulation of matrix metalloproteinase-2 and matrix metalloproteinase-9. Two human melanoma cell lines were used in this study: the SK-MEL-110, carrying a mutated p53 gene, and the SK-MEL-147, carrying the wild-type p53 gene. SK-MEL-110 cells infected with AdCMV.p53 exhibited decreased invasion capability from day 1 after infection, compared with cells not infected or infected with the control vector AdCMV.Null. This reduced invasiveness was associated with decreased matrix metalloproteinase-2 levels in conditioned media whereas no changes were detected in matrix metalloproteinase-9 secreted levels. No modulation in matrix metalloproteinase-2 mRNA levels was detectable, however, after wild-type p53 gene transfer. Furthermore, protein expression of secreted tissue inhibitor of metalloproteinase-2 was not altered by AdCMV.p53 treatment. In contrast, in SK-MEL-147 cells, AdCMV.p53 did not affect cell invasiveness and levels of secreted matrix metalloproteinase-2. Gene transfer of wild-type p53 inhibited proliferation of both cell lines, showing that also SK-MEL-147 cells respond to wild-type p53 overexpression. This novel mechanism of action of wild-type p53 gene transfer may contribute to its antitumor effect by downregulating cell invasion and matrix metalloproteinase-2 secreted levels in mutated p53 human melanoma cell lines.

Membrane-type 1 matrix metalloproteinase mRNA expression in colorectal cancer.

Membrane-type matrix metalloproteinases are recently described proteolytic enzymes belonging to the matrix metalloproteinase family. Initial studies have indicated that membrane-type matrix metalloproteinases are involved in tumor invasion and metastasis. Membrane-type 1 matrix metalloproteinase is the first membrane-type matrix metalloproteinase to be described. The aim of this study was to investigate the expression of membrane-type 1 matrix metalloproteinase mRNA in colorectal cancer. Samples were collected from surgical specimens of patients with colorectal adenocarcinoma and were immediately frozen in liquid nitrogen and stored at -80 degrees C until processed. Both normal and cancer tissue was taken from each patient. TNM stage, tumor differentiation, mucin production, and vascular invasion were assessed. Northern blotting was used to quantify membrane-type 1 matrix metalloproteinase mRNA levels in the samples using a membrane-type 1 matrix metalloproteinase cDNA clone. X-ray film images were digitized and densitometry was used to quantify bands. All samples were normalized against 18S rRNA levels. Results are expressed as the ratio of cancer to normal tissue levels. Statistical analysis was performed using analysis of variance, with P < 0.05 accepted as the level of significance. A total of 32 samples were prospectively analyzed. The correlation between TNM stage and increased expression of membrane-type 1 matrix metalloproteinase mRNA in cancer tissue over normal tissue is expressed in the mean ratio of cancer to normal tissue expression for Stages I through IV, respectively: 1.4 +/- 0.2 (12 patients); 4.1 +/- 2.6 (8 patients); 3.4 +/- 3 (7 patients); and 4.5 +/- 5 (5 patients). Stage I is significantly different from Stages II and IV (P < 0.05). These preliminary results show an overall increasing trend in membrane-type 1 matrix metalloproteinase expression with increasing tumor stage. However, there was no correlation between membrane-type 1 matrix metalloproteinase expression and mucin production, degree of tumor differentiation, or vascular invasion. Preliminary results indicate that membrane-type 1 matrix metalloproteinase levels correlate with increasing tumor stage.

Effects of culture conditions and exposure to catabolic stimulators (IL-1 and retinoic acid) on the expression of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs) by articular cartilage chondrocytes.

The chondrocytes of articular cartilage synthesize a number of proteinases which are capable of degrading the component molecules of this specialized extracellular matrix. The use of class-specific proteinase inhibitors indicates that major activities responsible for catabolism of proteoglycan (aggrecan) and collagen are attributable to zinc-dependent metalloproteinases. In this study, we have compared the mRNA expression profiles of two matrix metalloproteinases (MMP-3 and MMP-13) and five disintegrin-metalloproteinases (ADAM-10, ADAM-9, ADAM-15, TNF-alpha-converting enzyme and decysin) by chondrocytes (human, porcine and bovine) from fresh cartilage and in cartilage explant cultures and isolated cells cultured in monolayer or in agarose gels. Such cultures were maintained in the presence or absence of interleukin-1 (IL-1) or all-trans-retinoic acid, two agents which promote cartilage matrix degradation in vitro. Whereas transcripts for all metalloproteinases examined were detected in chondrocytes from human osteoarthritic cartilage in monolayer cultures, mRNAs for ADAM-15 and decysin were not present in fresh osteoarthritic human cartilage or explant cultures. Similarly, expression of porcine and bovine metalloproteinase mRNAs varied with different culture conditions. Novel cDNA sequences obtained for porcine and bovine MMP-3 and MMP-13, porcine ADAM-10, porcine and bovine ADAM-9 and porcine TACE confirmed expression of mRNAs for these molecules by articular chondrocytes. Quantitative RT-PCR analysis was used to determine the effects of IL-1 and retinoic acid on metalloproteinase mRNA levels in human chondrocytes cultured in monolayer and in porcine chondrocytes cultured in agarose. For the MMPs, IL-1 treatment resulted in an approximately two to threefold increase in human and porcine MMP-3 and MMP-13 mRNAs, while retinoic acid treatment caused a statistically significant increase in human MMP-3 mRNA levels, but no significant change in transcript levels for porcine MMP-3 nor human or porcine MMP-13. The mRNA levels for ADAM-15 were elevated in human monolayer chondrocytes exposed to IL-1 or retinoic acid, while transcripts levels for TNF-alpha converting enzyme were increased in response to retinoic acid. In contrast, ADAM-9 mRNA levels were decreased in human monolayer chondrocytes exposed to IL-1 or retinoic acid. The results demonstrate that chondrocyte metalloproteinase expression can vary dependent on cell environment in situ and in vitro, and information on chondrocyte MMP and ADAM gene expression following cytokine (IL-1) or retinoid stimulation.

DGLA discoordinately suppresses IL-1 induced metalloproteinase mRNA levels in human synovial fibroblasts.

DGLA discoordinately suppresses IL-1 induced metalloproteinase mRNA levels in human synovial fibroblasts.

Matrix Metalloproteinase Expression During Mouse Peri‐implantation Development

The purpose of this study was to define the temporal expression and to quantitate the mRNA levels of collagenase, 72 kDa, 92 kDa, and membrane-type matrix metalloproteinase during the peri-implantation period of pregnancy in the mouse uterus. Embryonic expression of 72 kDa and 92 kDa matrix metalloproteinases, as well as interleukin 1 alpha, was also investigated. Uterine matrix metalloproteinases were detected using gelatin substrate gel electrophoresis (zymography) and reverse-transcription polymerase chain reaction methodology was used to detect and quantitate different mRNA species in the mouse uterus and blastocyst. Collagenase, 72 kDa, and 92 kDa matrix metalloproteinases are developmentally regulated during the peri-implantation period of pregnancy, but membrane-type matrix metalloproteinase appears to be expressed constitutively. Matrix metalloproteinase mRNA levels have been quantitated and confirm the observed developmental expression patterns. Prominent expression of bot 92 kDa matrix metalloproteinase and interleukin 1 alpha was observed in blastocysts during outgrowth while weak expression of the 72 kDa matrix metalloproteinase was detected. The date provide evidence of matrix metalloproteinase expression in vivo and substantiate their potential role in tissue remodeling prior to and during blastocyst implantation. Expression of interleukin 1 alpha, 92 kDa, and 72 kDa matrix metalloproteinases suggests that these proteins are important for trophoblast invasion associated with implantation of the early embryo.