MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization.

As the only reliable treatment option for end-stage liver diseases, conventional liver transplantation confronts major supply limitations. Accordingly, the decellularization of discarded livers to produce bioscaffolds that support recellularization with progenitor/stem cells has emerged as a promising translational medicine approach. The success of this approach will substantially be determined by the extent of extracellular matrix (ECM) preservation during the decellularization process. Here, we assumed that the matrix metalloproteinase (MMP) inhibition could reduce the ECM damage during the whole liver decellularization of an animal model using a perfusion-based system. We demonstrated that the application of doxycycline as an MMP inhibitor led to significantly higher preservation of collagen, glycosaminoglycans, and hepatic growth factor (HGF) contents, as well as mechanical and structural features, including tensile strength, fiber integrity, and porosity. Notably, produced bioscaffolds were biocompatible and efficiently supported cell viability and proliferation in vitro. We also indicated that produced bioscaffolds efficiently supported HepG2 cell function upon seeding onto liver ECM discs using albumin and urea assay. Additionally, MMP inhibitor pretreated decellularized livers were more durable in contact with collagenase digestion compared to control bioscaffolds in vitro. Using zymography, we confirmed the underlying mechanism that results in these promising effects is through the inhibition of MMP2 and MMP9. Overall, we demonstrated a novel method based on MMP inhibition to ameliorate the ECM structure and composition preservation during liver decellularization as a critical step in fabricating transplantable bioengineered livers.

Expression and localization of the MMP inhibitor RECK in a rat model of COPD: its involvement in the development of COPD.

OBJECTIVE: To unravel the potential function of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as the matrix metalloproteinase (MMP) inhibitor in the development of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: Twelve specific pathogen-free Sprague Dawley rats were randomly assigned to the control cohort (n = 6) or the COPD cohort (n = 6). COPD model was developed by tobacco smoke exposure. Functional residual capacity (FRC), static lung compliance (Cchord), ratio of forced expiratory volume in 0.1 s to forced vital capacity (FEV0.1/FVC), and peak expiratory flow (PEF) were detected by respiratory function tests. Immunohistochemistry was performed to determine the pathological changes as well as the expression and localization of RECK in pulmonary tissue. RECK expression was further quantified by real-time polymerase chain reaction (PCR) and Western blot assays. RESULTS: COPD rats had significantly reduced FEV0.1/FVC% and PEF values but increased FRC and Cchord levels, as compared to the control cohort (p < 0.05). Hematoxylin and eosin (HE) staining indicated typical COPD pathological changes, including leukocyte infiltration, airway thickening, alveoli fusion, etc., in the COPD rats. IHC indicated reduced expression of RECK in the COPD cohort, which was mainly expressed on the epithelium and partly expressed on subepithelial cells and inflammatory cells. Real-time PCR and Western blot assays further revealed the significantly lower expression of RECK in lung tissue from the COPD cohort. CONCLUSIONS: RECK is mainly expressed on airway epithelial cells. COPD rats expressed significantly lower RECK levels, indicating that RECK exhibits a protective function in the development of COPD.

Effects of adrenocorticotrophic hormone on the expression of matrix metalloproteinases and their inhibitors in the bovine ovary.

Cattle undergo numerous environmental and management stressors that reduce fertility and affect ovulation. The extracellular matrix of the follicle wall can be altered by matrix metalloproteinases (MMPs), the activities of which are regulated by interleukins and tissue-specific inhibitors of metalloproteinases (TIMPs), especially during ovulation. The aims of the present study were to: (1) evaluate changes in the hormone milieu, the localisation and activity of MMP2 and MMP9 and the localisation of MMP14, TIMP1 and TIMP2 in response to adrenocorticotrophic hormone (ACTH) during the preovulatory period in cows; and (2) determine the direct effects of ACTH on the mRNA expression of MMP2 and MMP9 in the cultured follicle wall of bovine ovaries obtained from an abattoir. 100IU ACTH was administered during pro-oestrus every 12h until ovariectomy, which was performed before ovulation. Cortisol concentrations in the plasma and follicular fluid (FF) of preovulatory follicles were higher in ACTH-treated than control cows. Progesterone presented subluteal concentrations in plasma of ACTH-treated cows (P<0.05). MMP2 immunostaining and activity in ovaries were higher in ACTH-treated than control cows (P<0.05), whereas MMP9 immunostaining was similar between the two groups. However, unlike in control cows, MMP9 activity was absent in the FF of ACTH-treated cows. These results suggest that the administration of ACTH during the preovulatory period in cows could cause changes that culminate in modifications in the content and activation of MMPs and TIMPs in the ovary, which could interfere with the ovulation process.

Hierarchical virtual screening of the dual MMP-2/HDAC-6 inhibitors from natural products based on pharmacophore models and molecular docking.

The dual-target inhibitors tend to improve the response rate in treating tumors, comparing with the single-target inhibitors. Matrix metalloproteinase-2 (MMP-2) and histone deacetylase-6 (HDAC-6) are attractive targets for cancer therapy. In this study, the hierarchical virtual screening of dual MMP-2/HDAC-6 inhibitors from natural products is investigated. The pharmacophore model of MMP-2 inhibitors is built based on ligands, but the pharmacophore model of HDAC-6 inhibitors is built based on the experimental crystal structures of multiple receptor-ligand complexes. The reliability of these two pharmacophore models is validated subsequently. The hierarchical virtual screening, combining these two different pharmacophore models of MMP-2 and HDAC-6 inhibitors with molecular docking, is carried out to identify the dual MMP-2/HDAC-6 inhibitors from a database of natural products. The four potential dual MMP-2/HDAC-6 inhibitors of natural products, STOCK1 N-46177, STOCK1 N-52245, STOCK1 N-55477, and STOCK1 N-69706, are found. The studies of binding modes show that the screened four natural products can simultaneously well bind with the MMP-2 and HDAC-6 active sites by different kinds of interactions, to inhibit the MMP-2 and HDAC-6 activities. In addition, the ADMET properties of screened four natural products are assessed. These found dual MMP-2/HDAC-6 inhibitors of natural products could serve as the lead compounds for designing the new dual MMP-2/HDAC-6 inhibitors having higher biological activities by carrying out structural modifications and optimizations in the future studies.

Identification of Inhibitors of MMPS Enzymes via a Novel Computational Approach.

Matrix metalloproteases (MMPs) are a family of zinc-dependent proteinases that play complex and diverse roles in metabolism, which are vital for physiological development. In this paper, we present a novel method to identify peptide binding to seven matrix metalloproteases. First, we propose a novel sampling criteria for constructing a training set for each new peptide motif. Then, we select nine physicochemical properties of amino acids and compute their auto-cross covariance to effectively extract features for both natural and non-natural amino acids. Finally, we adopt random forest to predict binding values of each peptide motif respectively with seven MMPs. Our method verifies on 1300 known peptide motifs binding to seven MMPs and achieved preeminent Pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) on all seven MMPs, especially of 0.9181 and 9.3827 on MMP-7. We predict binding values of 4000 peptide motifs and identify peptides preferentially bind to MMP-2 and MMP-7. We herein report 4 novel inhibitor candidates of Asp-Ile-Phe, Asp-Ile-Tyr, Asp-Ile-Lys and Hser-Gly-Phe with high potency and selectivity binding to MMP-2, as well as 6 novel inhibitor candidates of Chg-Ile-Ile, Chg-Ile-Leu, Chg-Ile-Glu, Chg-Ile-Met, Chg-Val-Ile and Chg-Val-Leu selectively binding to MMP-7. Our findings facilitate the identification of inhibitors with good potency as well as desirable selectivity, providing significant insights of candidate inhibitor drugs.

In vivo wound-healing activity of Euphorbia characias subsp. wulfenii: Isolation and quantification of quercetin glycosides as bioactive compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: The latex and the aerial parts of Euphorbia characias L. (Euphorbiaceae) have been used as medicinal plant to treat wounds and warts in traditional medicine. AIM OF THE STUDY: The effect of the plant extract was tested in vivo and in vitro with experimental models to find scientific evidence for traditional use in wound healing. Potentially active wound-healer compounds were isolated from the active fraction using fractionation procedures under the guidance of biological assay and the possible role of the compounds in the wound healing process was also determined. MATERIAL AND METHODS: N-hexane, ethyl acetate, and methanol extracts were successively prepared from the aerial parts of E. characias subsp. wulfenii. The extracts were tested with linear incision, circular excision wound models and the hydroxyproline assay method to assess the wound-healing activity. The inhibition of the increase in capillary permeability induced by acetic acid, an acute inflammation model, was used to assay the anti-inflammatory activity. Different chromatographic separation techniques on sephadex and silica gel columns, and bioassay guided assay techniques have been used to isolate the active compounds of the plant. Moreover, hyaluronidase, collagenase and elastase enzymes inhibitory effect of active principle were investigated in vitro to find out the mechanism of action. RESULTS: The methanol (MeOH-ex) extract of the aerial parts of E. characias subsp. wulfenii showed significant wound healing activity (linear incision wound model: 43.04%; circular excision wound model 65.24%) and anti-inflammatory activity (34.74%). The methanol extract was separated into its fractions by column chromatography for isolation of efficient compounds. Biological activity of the fractions were assessed and further isolation and purification processes have been carried out in the active fraction. Isolation studies were carried out from the MeOH-ex fraction to obtain active constituents and their structures were elucidated to be quercetin-3-O-rhamnoside (quercitrin), quercetin-3-O-galactoside (hyperoside), and quercetin-3-O-arabinoside (guaijaverin). Further in vitro and in vivo assays showed that quercetin derivatives were responsible for the wound-healing activity of the plant, and also found to be significant anti-elastase and anti-collagenase activities. The amounts of three compounds, isolated from active fraction, were determined by using high performance liquid chromatography. Calibration equation was calculated with dilutions, prepared from pure substances, and assay was performed in total extract, prepared from E. characias subsp. wulfenii. It was detected that the plant had 1.22% quercitrin, 0.35% hyperoside, and 0.11% guaijaverin. The validation of the analytical method was performed by linearity, precision, limit of detection, and limit of quantification parameters. CONCLUSION: Present study supported the traditional use of the aerial parts E. characias subsp. wulfenii as wound healer and quercetin derivatives were isolated as active components from the active fraction by using bioassay-guided fractionation technique.

Thai plants with high antioxidant levels, free radical scavenging activity, anti-tyrosinase and anti-collagenase activity.

BACKGROUND: Ultraviolet radiation from sunlight induces overproduction of reactive oxygen species (ROS) resulting in skin photoaging and hyperpigmentation disorders. Novel whitening and anti-wrinkle compounds from natural products have recently become of increasing interest. The purpose of this study was to find products that reduce ROS in 14 Thai plant extracts. METHODS: To determine total phenolic and flavonoid content, antioxidant activity, anti-tyrosinase activity and anti-collagenase activity, we compared extracts of 14 Thai plants prepared using different solvents (petroleum ether, dichloromethane and ethanol). Antioxidant activities were determined by DPPH and ABTS assays. RESULTS: Total phenolic content of the 14 Thai plants extracts was found at the highest levels in ethanol followed by dichloromethane and petroleum ether extracts, respectively, while flavonoid content was normally found in the dichloromethane fraction. Scavenging activity ranged from 7 to 99% scavenging as assessed by DPPH and ABTS assays. The ethanol leaf extract of Ardisia elliptica Thunb. had the highest phenolic content, antioxidant activity and collagenase inhibition, while Cassia alata (L.) Roxb. extract had the richest flavonoid content. Interestingly, three plants extracts, which were the ethanolic fractions of Annona squamosa L., Ardisia elliptica Thunb. and Senna alata (L.) Roxb., had high antioxidant content and activity, and significantly inhibited both tyrosinase and collagenase. CONCLUSION: Our finding show that the ethanol fractions of Annona squamosa L., Ardisia elliptica Thunb. and Senna alata (L.) Roxb. show promise as potential ingredients for cosmetic products such as anti-wrinkle agents and skin whitening products.

Expression of TIMP-2 in HPV-16 infected oral squamous cell carcinoma in patients in Iraq

Aim: To determine the expression of tissue inhibitors of metalloproteinases (TIMP-2) in oralsquamous cell carcinoma (OSCC) and the difference in its expression level between positiveand negative HPV-16 (human papilloma virus- 16) OSCC patients. Methods: This study wasconducted on 33 biopsies obtained from patients with OSCC and 10 normal oral mucosa ascontrols. In situ hybridization (ISH) was used to investigate the presence of HPV-16, whileimmunohistochemistry (IHC) was used to estimate the expression level of TIMP-2. Results: TheTIMP-2 was expressed in 27 (81.8%) of OSCC sections with no significant difference betweenits expression level in HPV-16 positive and HPV-16 negative OSCC cases (p=0.058). TIMP-2was found to be highly expressed in OSCC sections, and the presence of HPV was not relatedto its overexpression. Conclusions: The percentage of samples that appeared to accommodatedetectable HPV-16 was high, but no significant difference was observed in relation to TIMP-2expression level. Future studies with a larger number of patients are highly recommended toaddress the possible association between TIMp-2 and OSCC positive HPV-16.

[The utility of metalloproteinases (MMPs) and their inhibitors (TIMPs) in diagnostics of gynecological malignancies]. / Przydatnosc oznaczania metaloproteinaz (MMPs) i ich inhibitorów (TIMPs) w diagnostyce nowotworów narzadu rodnego.

Metalloproteinases (MMPs) are a family of proteolytic enzymes, involved in the degradation of collagen and other extracellular matrix components. They play a very important role in many physiological processes, i.e. angiogenesis, hemostasis, cyclic changes in the endometrium, wounds healing, as well as in tumor growth and spreading. Already performed studies have shown significant increase in the expression of MMP-2 and MMP-9 in the most common gynecological cancer (cervix, endometrium, ovary) compared to normal tissue and benign lesions. In addition, the MMP-9 concentration correlated with the clinical stage and the presence of distant metastases. Moreover the level of MMP-2 was significantly associated with the degree of malignancy. MMP-7 may be helpful in the diagnosis of ovarian cancer and useful in estimating of lymph node metastasis presence in endometrial cancer. In the detection of cervical cancer it may be useful to evaluate the expression of MMP-11 and MMP-12 (absent in normal cells) and their increase according to the degree of tissue damage. The usefulness of metalloproteinases in the diagnosis of gynecological cancer still requires confirmation test. However, it appears that they will be valuable factors in diagnostic complement, especially in combination with conventional markers, i.e. CA 125, SCCAg or HE-4.

Systems pharmacology-based approach for dissecting the active ingredients and potential targets of the Chinese herbal Bufei Jianpi formula for the treatment of COPD.

BACKGROUND: The Chinese herbal Bufei Jianpi formula (BJF) provides an effective treatment option for chronic obstructive pulmonary disease (COPD). However, the systems-level mechanism underlying the clinical effects of BJF on COPD remains unknown. METHODS: In this study, a systems pharmacology model based on absorption filtering, network targeting, and systems analyses was applied specifically to clarify the active compounds and therapeutic mechanisms of BJF. Then, a rat model of cigarette smoke- and bacterial infection-induced COPD was used to investigate the therapeutic mechanisms of BJF on COPD and its comorbidity. RESULTS: The pharmacological system successfully identified 145 bioactive ingredients from BJF and revealed 175 potential targets. There was a significant target overlap between the herbal constituents of BJF. These results suggested that each herb of BJF connected with similar multitargets, indicating potential synergistic effects among them. The integrated target-disease network showed that BJF probably was efficient for the treatment of not only respiratory tract diseases but also other diseases, such as nervous system and cardiovascular diseases. The possible mechanisms of action of BJF were related to activation of inflammatory response, immune responses, and matrix metalloproteinases, among others. Furthermore, we demonstrated that BJF treatment could effectively prevent COPD and its comorbidities, such as ventricular hypertrophy, by inhibition of inflammatory cytokine production, matrix metalloproteinases expression, and other cytokine production in vivo. CONCLUSION: This study using the systems pharmacology method, in combination with in vivo experiments, helped us successfully dissect the molecular mechanism of BJF for the treatment of COPD and predict the potential targets of the multicomponent BJF, which provides a new approach to illustrate the synergetic mechanism of the complex prescription and discover more effective drugs against COPD.

Aplicação de modelagem molecular e de formalismo do CAMD (Computer-Aided Molecular Design) na elucidação do mecanismo de ação de inibidores de metalopropteinases de matriz / Molecular modeling methods and computer-aided molecular design (CAMD) formalisms for elucidating the mechanism of action of matrix metalloproteinases inhibitors

As metaloproteinases de matriz (MMP) são enzimas superexpressas em quase todos os tumores humanos, sendo que os subtipos MMP-2 e MMP-9 têm sido associados ao potencial metastático e prognóstico desfavorável em neoplasias malignas como, por exemplo, melanoma metastático e glioma. Compostos capazes de inibir a atividade destas enzimas podem representar potenciais agentes terapêuticos. O composto 4-nerolidilcatecol (4-NC), isolado de plantas do gênero Pothomorphe, apresentou resultados promissores para o tratamento do melanoma e glioma e foi capaz de atuar em várias etapas bioquímicas importantes envolvidas na progressão dessas patologias, inclusive inibindo MMP-2 e MMP-9. No entanto, o mecanismo de ação do 4-NC não está completamente elucidado. O presente estudo envolveu a aplicação de métodos de modelagem molecular e de formalismos do planejamento de novas moléculas auxiliado por computador, CAMD (Computer-Aided Molecular Design) a fim de explorar a interação entre esta molécula e as enzimas MMP-2 e MMP-9, além de planejar novos inibidores para estes alvos. Análise exploratória de dados, que compreende a análise de agrupamentos hierárquicos e de componentes principais. foi desenvolvida para um conjunto de hidroxamatos (N=64) descritos como inibidores de MMP-2 e MMP-9, a fim de identificar as propriedades moleculares que mais influenciavam o processo de discriminação dos compostos. As propriedades termodinâmicas, eletrônicas e estéricas foram importantes para descrever os compostos mais ativos no conjunto de dados da MMP-2. Para a MMP-9, o coeficiente de distribuição (ClogD) em pH 1,5 foi relevante no processo de discriminação do conjunto. A presença de substituintes volumosos na porção R3 parece ser crucial para o conjunto de inibidores investigados. Esta região está envolvida em interações moleculares com a cavidade S1 de ambas as enzimas, mas há um limite de volume a ser considerado para estes substituintes. O formalismo QSAR-4D independente do receptor (IR) foi aplicado ao mesmo conjunto de dados e permitiu estabelecer o mapeamento do farmacóforo, além de explorar diferentes alinhamentos para a obtenção da hipótese de conformação bioativa prevista pelo melhor modelo de QSAR. OS modelos QSAR apresentaram boa capacidade de previsão, auxiliaram na proposição de novos inibidores e estimaram a atividade do 4-NC. Com o melhor modelo QSAR para MMP-9 (N=64), a atividade prevista para o 4-NC foi classificada na faixa dos inibidores com atividade moderada. Entretanto, o melhor modelo QSAR obtido para MMP-2 (N=38) não foi capaz de prever, de forma adequada, a atividade de compostos com arcabouço químico diferente daqueles utilizados na construção dos modelos. Estudos de ancoramento molecular foram desenvolvidos para investigar a orientação do 4-NC no sitio catalítico das duas enzimas e as interações que poderiam ser estabelecidas nestes complexos. Duas conformações favoráveis foram encontradas. Simulações computacionais de dinâmica molecular foram desenvolvidas com os complexos mais promissores selecionados nos estudos de ancoramento, a fim de obter informações mais detalhadas e de maior confiabilidade. sobre suas interações intermoleculares. O 4-NC tende a se orientar no sítio de forma a acomodar sua cadeia lateral no bolso S1 adjacente ao sítio catalítico em ambas as enzimas. Ensaios de zimografia também foram realizados com o objetivo de elucidar possíveis contribuições da cadeia lateral e do núcleo catecólico do 4-NC na atividade inibitória frente às enzimas em estudo. O núcleo catecólico parece ser o responsável por sua atividade, pois o composto 1,2dimetoxibenzeno, que possui as hidroxilas bloqueadas por grupos metil, não foi capaz de exercer atividade inibitória significante frente à MMP-2 e MMP-9. Estudos de voltametria reforçaram a hipótese de que o 4-NC tem a capacidade de quelar os íons zinco presentes no tampão de incubação

Matrix metalloproteinases (MMP) enzymes are overexpressed in almost all human tumors, and MMP-2 and MMP-9 subtypes have been associated with metastatic potential and poor prognosis in malignant tumors, such as metastatic melanoma and glioma. Compounds capable of inhibiting the activity of theses enzymes would be considered as potential therapeutic agents. The 4-nerolidylcatechol compound (4-NC), isolated from plants of genus Pothomorphe, has showed promising results in the treatment of melanoma and glioma, and was able to act in several important biochemical steps involved in the progression of these diseases, as well as inhibiting MMP-2 and MMP-9. However, the 4-NC mechanism of action is not completely understood. This study has involved the application of molecular modeling methods and formalisms of computer-aided molecular design (CAMD) in order to explore the interaction between 4-NC and MMP-2/MMP-9, and to design new inhibitors for these targets. Exploratory data analysis, which comprises hierarchical cluster analysis and principal components analysis, was performed to a set of hydroxamates (N=64). previously reported as MMP-2 and MMP-9 inhibitors, in order lo identify the molecular properties that is most critical for the discrimination process regarding the investigated compounds. The thermodynamic, electronic, and steric properties were: quite important to describe the highly active compounds in the data set of MMP-2, whereas the apparent partition coefficient (ClogD) at pH 1.5 was the property more relevant for MMP-9 data set. The presence of bulky substituents on the R3 moiety seems to be crucial for this set of inhibitors due to the molecular interaction with the S1 subsite of both enzymes. However, there is a limit regarding the substituents volume in this region. Receptor independent (RI) 4D-QSAR analysis was applied lo the same data set and it was possible to establish the pharmacophore mapping, besides to explore different alignments in order to generate the hypothesized bioactive conformation through the best QSAR model. The QSAR models have presented good predictability, assisted in proposing new inhibitors, and estimated the activity of 4-NC. Regarding the best QSAR model for MMP-9 (N=64), the 4-NC predicted activity was classified in the range of the moderate active inhibitors. The best QSAR model obtained for MMP-2 (N=38), however was not able to properly predict the activity for compounds with different chemical scaffold from those used to build up the QSAR model. Molecular docking studies have been developed to investigate the 4-NC binding mode into the catalytic site of the two enzymes and the interactions that could be established in those complexes. The results have shown two favorable conformers regarding the MMP inhibition. Molecular dynamics computational simulation were combined to molecular docking studies in order to obtain more detailed and reliable information regarding the intermolecular interactions of each complex. The 4-NC molecule tends to accommodate the side chain in the S1 pocket adjacent to the catalytic site in both enzymes. Experimental zymography assays were also performed to elucidate the possible contribution of the side chain and the catechol core in the 4-NC inhibitory activity against the MMP-2 and MMP-9 enzymes. The catechol core seems to be responsible for its activity, since the 1,2 dimethoxybenzene compound, which has the hydroxyl blocked by a methyl group, was not able to exert any significant inhibition on enzymes. Voltametric assays confirmed the hypothesis that 4-NC chelates zinc ions present in the incubation buffer

Expression of matrix metalloproteinases (MMP-2, MMP-9) and their inhibitors (TIMP-1, TIMP-2) in canine testis, epididymis and semen.

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP-2 and MMP-2 activity in high than low sperm concentration samples (p < 0.001). ProMMP-9 and MMP-9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP-2 and MMP-2 were associated with high sperm motility (≥70%, p < 0.001). Sperm-rich fraction showed significantly (eight-fold) higher proMMP-9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP-2-specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP-9, TIMP-1 and TIMP-2 were absent in these cells. Matrix metalloproteinase-9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP-1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP-2 localization along acrosomal region of sperm, while MMP-9, TIMP-1 and TIMP-2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP-2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP-2 and MMP-9 may serve as an alternative biomarker in determining semen quality.

Effect of psychological stress on the structure of the temporomandibular joint and the expression of MMP-3 and TIMP-3 in the cartilage in rats.

Our aim was to observe the effects of psychological stress on the structure of the temporomandibular joint (TMJ), and to evaluate the expression of matrix metallopeptidase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in condylar chondrocytes in rats. The rats were divided into 3 groups of 12 according to the duration of psychological stress: 3 weeks or 6 weeks, and 6 weeks of recovery. A fourth group of 12 rats was used as controls. Each rat was evaluated by the open-field test and the weight measured. The results confirmed psychological stress in 24 of the 36 rats (67%). The tissues of the TMJ were stained with haematoxylin and eosin and pathological changes were studied under a light microscope. MMP-3 and TIMP-3 expression was investigated using the SP kit. The experimental groups showed thinning of articular cartilage, shedding of collagen fibres, cracks in the articular discs, and other structural changes that were aggravated with time, from three weeks to six weeks. The 6-week recovery group showed an improvement in these changes, which indicated the initiation of joint repair. The MMP-3 expression rate correlated with the degree of joint lesion, while the TIMP-3 rate showed an opposite trend and was highest in the 6-week recovery group. Our findings clearly indicate that psychological stress may play an important part in the development of TMJ diseases in rats; further studies should be made to extrapolate the results to other models before clinical use.

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and ß.

Zinc metalloproteinases meprin α and meprin ß are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin α and meprin ß and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin ß. Kinetic studies revealed competitive (PPNDS) and mixed competitive/noncompetitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin ß active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin ß reported to the date. These results demonstrate the ability of meprin α and ß assays to identify selective compounds and discard artifacts of primary screening.

DENTINE CARIES: ACID-TOLERANT MICROORGANISMS AND ASPECTS ON COLLAGEN DEGRADATION.

Dental caries is a common disease all over the world, despite the fact that it can be both effectively prevented and treated. It is driven by acids produced by oral microorganisms as a consequence of their metabolism of dietary carbohydrates. Given enough acid challenge, eventually the tooth enamel barrier will be broken down, and the carious lesion will extend into underlying hard tissue, forming a macroscopic cavity in the dentine. In comparison to biofilm on enamel, a dentine carious lesion provides a vastly different environment for the residing microorganisms. The environment influences the types and numbers of microorganisms that can colonize the dentine caries lesion. The overall aims for this thesis are to enumerate and further study microorganisms found in established dentine caries lesions and also to illuminate how host-derived proteolytic enzymes might contribute to this degradation, not only to better understand the caries process in dentine but also to find incitements for new methods to influence the natural progression of caries lesions. In Paper I, the numbers of remaining viable microorganisms after completed excavation using two excavation methods were investigated. Samples of carious dentine tissue were collected before and after excavation and cultivated on different agar media in different atmospheres. Analysis was performed by counting the number of colony-forming units (CFUs). Key findings: The number of remaining microorganisms after excavation was low for both methods, but some microorganisms always remained in the cavity floors even when the cavities were judged as caries free using normal clinical criteria. In Paper II, the acid tolerant microbiota in established dentine caries lesions was investigated. Samples were taken as in Paper I, but on three levels (superficial, center of lesion, floor of lesion after completed excavation). The samples were cultivated in anaerobic conditions on solid pH-selective agar media of different acidity. Key findings: Each investigated lesion harbored a unique microbiota in terms of both species composition and numbers of microorganisms. This indicates that various combinations of aciduric microorganisms can colonize, survive in and probably also propagate dentine carious lesions. We also found that solid pH-selective agars can be used successfully to select acid-tolerant microorganisms in caries lesions. This would preserve their phenotypic traits for further study. In Paper III, the relation between salivary levels of matrix metalloproteinase-8 (MMP-8), salivary levels of tissue inhibitor of MMP (TIMP-1), and the presence of manifest caries lesions in a large number of subjects was investigated. Saliva samples were collected and analyzed for concentrations of MMP-8, TIMP-1 and total protein using immunofluorometric assays, enzyme linked immunosorbent assays and Bradford assays, respectively. Key findings: Subjects with manifest caries lesions had significantly elevated levels of salivary MMP-8 compared to subjects without caries lesions. TIMP-1 was not significant in any case. In Paper IV, a new method for generating bioactive demineralized dentine matrix substrate (DDM) was developed using a dialysis system and two different demineralization approaches (acetic acid or EDTA). The generated DDM was subsequently analyzed for the presence of type 1 collagen, active MMP-8 and hydroxyproline (HYP) levels using SDS-PAGE, ELISA or immunofluorescence assay. Key findings: Both demineralization methods produced a substrate rich in collagen and with preserved MMP-8 activity. This report presents new knowledge on the composition of the acid tolerant dentine caries microbiota from three levels in dentine carious lesions and on the efficacy of operative caries removal on the numbers of viable microorganisms in the caries free cavity using two operative methods. Moreover, the basic mechanisms behind collagen degradation in the dentine caries process are studied from both a clinical and laboratory perspective. The report also provides a reference for further studies on dentine caries microbiology and dentine caries collagen degradation mechanisms, both of which are known only in part.

Human gingival fibroblasts function is stimulated on machined hydrided titanium zirconium dental implants.

OBJECTIVES: The aim of this study was to evaluate the influence of different titanium zirconium (TiZr) alloy surfaces on primary human gingival fibroblasts (HGF) for improved soft tissue integration of dental implants. METHODS: TiZr polished, machined and machined+HCl/H2SO4 acid-etched surfaces were modified by cathodic polarization and/or HNO3/HF acid etching. Contact angle of surfaces was measured. The influence of modified TiZr surfaces on HGF was evaluated through the analysis of cell number, morphology, recovery after a wound (wound healing assay) and the expression of several genes, including matrix metalloproteinase-1 (MMP1) and metallopeptidase inhibitor-1 (TIMP1). RESULTS: Modification of TiZr surfaces decreased its hydrophilicity. Hydride implementation on TiZr surfaces via cathodic polarization increased TIMP1 expression and decreased MMP1/TIMP1 mRNA ratio. Cathodic polarization of machined surfaces promoted cell attachment. Cells on machined and machined+cathodic polarization surfaces grew aligned to the microgrooves whereas on all polished surfaces they grew randomly. Acid etching of polished and machined surfaces did not improve HGF function. CONCLUSIONS: Hydride implementation on TiZr machined surfaces may be used as new dental implant material for improved soft tissue integration. CLINICAL SIGNIFICANCE: Enhancing dental implant surfaces' bioactivity by hydride implementation may promote soft tissue attachment and sealing around the implant and reduce peri-implantitis related to ECM-destruction compared with conventional machined surfaces.

Matrix metalloproteinase, hyaluronidase and elastase inhibitory potential of standardized extract of Centella asiatica.

CONTEXT: Centella asiatica (L.) Urban (Apiaceae), a valuable herb described in Ayurveda, is used in the indigenous system of medicine as a tonic to treat skin diseases. OBJECTIVE: Centella asiatica methanol extract and its ethyl acetate, n-butanol and aqueous fraction, were subjected for the evaluation of skin care potential through the in vitro hyaluronidase, elastase and matrix metalloproteinase-1 (MMP-1) inhibitory assay. MATERIALS AND METHODS: The C. asiatica plant was extracted with methanol and fractionated with ethyl acetate, n-butanol and water. The enzymatic activities were evaluated using ursolic acid and oleanolic acid as standards. Isolate molecule asiaticoside was quantified in the crude extract and fractions through high-performance liquid chromatography (HPLC) and structural was characterized by liquid chromatography-mass spectroscopy (LC-MS) and ¹H nuclear magnetic resonance (NMR). Isolated compound was also evaluated for in vitro enzyme assays. RESULTS: Extract exhibited anti-hyaluronidase and anti-elastase activity with IC50 of 19.27 ± 0.37 and 14.54 ± 0.39 µg/mL, respectively, as compared to ursolic acid. Centella asiatica n-butanol fraction (CAnB) and isolated compound showed significant hyaluronidase (IC50 = 27.00 ± 0.43 and 18.63 ± 0.33 µg/mL) and elastase (IC50 = 29.15 ± 0.31 and 19.45 ± 0.25 µg/mL) inhibitory activities, respectively, and also showed significant MMP-1 inhibition (p < 0.05 and p < 0.01). DISCUSSION AND CONCLUSION: n-Butanol fraction was found to be most effective among the all fractions from which asiaticoside was isolated and further quantified by HPLC. This work concludes that the asiaticoside from C. asiatica may be a prospective agent for skin care.

Peri-implant versus periodontal wound healing.

AIM: Peri-implant gingival healing following one-stage implant placement was investigated and compared to periodontal healing. METHODS: Healing at surgical sites [implant (I) and adjacent teeth (T+)] was compared to non-operated tooth (T-) in non-smokers receiving one-stage implant. Periodontal Indices (PI, GI) were recorded at surgery and up to 12 weeks post-operatively. Peri-implant (PICF) and gingival crevicular fluids (GCF) were analysed for cytokines, collagenases and inhibitors. Data were analysed by linear mixed model regression analysis and repeated measures anova. RESULTS: Forty patients (22 females; 21-74 years old) completed the study. Surgical site GI, increased at week 1, decreased significantly during early healing (weeks 1-3; p = 0.0003) and continually decreased during late healing (weeks 6-12) for I (p < 0.01). PICF volume decreased threefold by week 12 (p = 0.0003). IL-6, IL-8, MIP-1ß and TIMP-1 levels significantly increased at surgical sites at week one, significantly decreasing thereafter (p < 0.016). Week one IL-6, IL-8 and MIP-1ß levels were ~threefold higher and TIMP-1 levels 63% higher, at I compared to T+ (p = 0.001). CONCLUSION: Peri-implant gingival healing, as determined by crevicular fluid molecular composition, differs from periodontal healing. The observed differences suggest that peri-implant tissues, compared to periodontal tissues, represent a higher pro-inflammatory state.

On-line screening of matrix metalloproteinase inhibitors by capillary electrophoresis coupled to ESI mass spectrometry.

Capillary electrophoresis (CE) with the use of mass spectrometry (MS) has been considered as a unique tool for microscale enzyme assay and inhibitor screening. In this study, matrix metalloproteinase-9 (MMP-9) was selected as target enzyme due to its important role in tumor invasion and metastasis. In order to define the optimal MS parameters, a two level half fraction factorial experimental design was performed. A background electrolyte consisting of 20mM ammonium acetate (pH 6.8) and a sheath liquid of water-methanol (50:50, v/v) containing 0.05% formic acid at a flow rate of 4µl/min were selected. This system was operated in the positive ion mode with a detection-limit of 10nM for the MMP reaction product and provided 60 folds enhancement of sensitivity by using selected reaction monitoring detection compared with MS full scan mode, which significantly increased the detectability of the system and therefore reduced the enzyme reaction time in both off-line and in-line mode. Both electrophoretically mediated microanalysis and pressure mediated microanalysis combined with MS detection were investigated for MMP inhibitor screening. Good repeatability (RSD of peak area and migration time were lower than 5.0%) and linearity (R(2)>0.996) were obtained for both in-capillary approaches. Several tetracycline antibiotics and natural products were selected to test the system. The results indicated an agreement on the ranking of inhibitory potency for both in-capillary approaches.

Water-soluble mmp-9 inhibitor prodrug generates active metabolites that cross the blood-brain barrier.

MMP-9 plays a detrimental role in the pathology of several neurological diseases and, thus, represents an important target for intervention. The water-soluble prodrug ND-478 is hydrolyzed to the active MMP-9 inhibitor ND-322, which in turn is N-acetylated to the even more potent metabolite ND-364. We used a sensitive bioanalytical method based on ultraperformance liquid chromatography with multiple-reaction monitoring detection to measure levels of ND-478, ND-322, and ND-364 in plasma and brain after administration of ND-478 and the metabolites. ND-478 did not cross the blood-brain barrier, as was expected; however the active metabolites ND-322 and ND-364 distributed to the brain. The active compound after administration of either ND-478 or ND-322 is likely ND-364. ND-322 is N-acetylated in both brain and liver, but it is so metabolized preferentially in liver. Since N-acetyltransferases involved in the metabolism of ND-322 to ND-364 are polymorphic, direct administration of the N-acetylated ND-364 would achieve the requisite therapeutic levels in the brain.