Effects of statins on matrix metalloproteinases and their endogenous inhibitors in human endothelial cells

Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 mu M) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.

Abnormal extracellular matrix protein transport associated with increased apoptosis of vascular smooth muscle cells in Marfan syndrome and bicuspid aortic valve thoracic aortic aneurysm

Background - Marfan syndrome (MS) is a genetic disorder caused by a mutation in the fibrillin gene FBN1. Bicuspid aortic valve (BAV) is a congenital heart malformation of unknown cause. Both conditions are associated with ascending aortic aneurysm and premature death. This study examined the relationship among the secretion of extracellular matrix proteins fibrillin, fibronectin, tenascin, and vascular smooth muscle cell (VSMC) apoptosis. The role of matrix metalloproteinase (MMP)- 2 in VSMC apoptosis was studied in MS aneurysm. Methods and Results - Aneurysm tissue was obtained from patients undergoing surgery ( MS: 4 M, 1 F, age 27 - 45 years; BAV: 3 M, 2 F, age 28 - 65 years). Normal aorta from subjects with nonaneurysm disease was also collected ( 4 M, 1 F, age 23 - 93 years). MS and BAV aneurysm histology showed areas of cystic medial necrosis (CMN) without inflammatory infiltrate. Immunohistochemical study of cultured MS and BAV VSMC showed intracellular accumulation and reduction of extracellular distribution of fibrillin, fibronectin, and tenascin. Western blot showed no increase in expression of fibrillin, fibronectin, or tenascin in MS or BAV VSMC and increased expression of MMP-2 in MS VSMCs. There was 4-fold increase in loss of cultured VSMC incubated in serum-free medium for 24 hours in both MS ( 27 +/- 8%) and BAV ( 32 +/- 14%) compared with control ( 7 +/- 5%). Conclusions - In MS and BAV there is alteration in both the amount and quality of secreted proteins and an increased degree of VSMC apoptosis. Up-regulation of MMP-2 might play a role in VSMC apoptosis in MS VSMC. The findings suggest the presence of a fundamental cellular abnormality in BAV thoracic aorta, possibly of genetic origin.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Résumant mon travail de thèse, l'article qui suit décrit un nouveau modèle animal servant à étudier l'impact combiné d'une ventilation mécanique (VM), d'une oxygénothérapie et d'une inflammation sur des poumons immatures. Cette étude permet, pour la première fois, de mesurer l'expression de gènes à distance d'une VM pour en analyser la cinétique. La VM représente un traitement intégral dans la prise en charge de prématurés. Sauvant des vies, elle est cependant non-physiologique et décrite comme nocive à court et à long terme, empêchant le bon développement pulmonaire. Nombreuses études se sont intéressées à l'impact immédiat de la VM sur les poumons, mais il n'existe à ce jour aucun modèle de rongeur pour en analyser les effets tardifs. Par analogie avec la clinique, nous avons créé un modèle avec un animal dont le stade développemental pulmonaire est comparable aux prématurés humains et consistant en une oxygénothérapie, une VM modérée avec intubation non chirurgicale, similaire à la pratique quotidienne, et un contexte inflammatoire mimant celui de chorioamnionite dans lequel bien des prématurés naissent. Nous avons ensuite réalisé une extubation pour permettre une période de rétablissement, puis fait des analyses et sur le plan structurel par histologie conventionnelle et en 3D, et sur le plan biologique, par analyse de l'expression de gènes et de protéines. Ce travail a permis de valider ce nouveau modèle comme outil de recherche pour réaliser des mesures à distance d'une VM chez des rats nouveau-nés. Comparant ces mesures à celles prises à la fin de la VM, nous observons: une augmentation initiale et transitoire des médiateurs impliqués dans la cascade inflammatoire dont le corrélat histologique est une maladie inflammatoire pulmonaire et, tardivement, une altération plus développementale de la structure pulmonaire avec diminution de l'alvéolarisation. Ceci pourrait être en partie dû à une expression asynchrone de gènes décrits comme importants pour la formation des alvéoles (matrix metalloproteinase 9, elastine). Offrant une nouvelle approche pour la recherche pulmonaire chez les rongeurs, ce modèle servira comme futur outil pour approfondir nos connaissances de la physiopathologie conduisant aux altérations structurelles retrouvées dans les poumons d'anciens prématurés soumis à une VM (dysplasie broncho-pulmonaire), pour tester l'influence de certains traitements (p.ex. surfactant) et pour étudier les effets de la VM en l'appliquant à des modèles transgéniques.

Expression of extracellular matrix components and their receptors in the central nervous system during experimental Toxoplasma gondii and Trypanosoma cruzi infection

Alterations in extracellular matrix (ECM) expression in the central nervous system (CNS) usually associated with inflammatory lesions have been described in several pathological situations including neuroblastoma and demyelinating diseases. The participation of fibronectin (FN) and its receptor, the VLA-4 molecule, in the migration of inflammatory cells into the CNS has been proposed. In Trypanosoma cruzi infection encephalitis occurs during the acute phase, whereas in Toxoplasma infection encephalitis is a chronic persisting process. In immunocompromised individuals such as AIDS patients, T. cruzi or T. gondii infection can lead to severe CNS damage. At the moment, there are no data available regarding the molecules involved in the entrance of inflammatory cells into the CNS during parasitic encephalitis. Herein, we characterized the expression of the ECM components FN and laminin (LN) and their receptors in the CNS of T. gondii- and T. cruzi-infected mice. An increased expression of FN and LN was detected in the meninges, leptomeninges, choroid plexus and basal lamina of blood vessels. A fine FN network was observed involving T. gondii-free and T. gondii-containing inflammatory infiltrates. Moreover, perivascular spaces presenting a FN-containing filamentous network filled with a4+ and a5+ cells were observed. Although an increased expression of LN was detected in the basal lamina of blood vessels, the CNS inflammatory cells were a6-negative. Taken together, our results suggest that FN and its receptors VLA-4 and VLA-5 might be involved in the entrance, migration and retention of inflammatory cells into the CNS during parasitic infections.

Functions of the extracellular matrix and matrix degrading proteases during tumor progression

Cell interactions with extracellular matrices are important to pathological changes that occur during cell transformation and tumorigenesis. Several extracellular matrix proteins including fibronectin, thrombospondin-1, laminin, SPARC, and osteopontin have been suggested to modulate tumor phenotype by affecting cell migration, survival, or angiogenesis. Likewise, proteases including the matrix metalloproteinases (MMPs) are understood to not only facilitate migration of cells by degradation of matrices, but also to affect tumor formation and growth. We have recently demonstrated an in vivo role for the RGD-containing protein, osteopontin, during tumor progression, and found evidence for distinct functions in the host versus the tumor cells. Because of the compartmentalization and temporal regulation of MMP expression, it is likely that MMPs may also function dually in host stroma and the tumor cell. In addition, an important function of proteases appears to be not only degradation, but also cleavage of matrix proteins to generate functionally distinct fragments based on receptor binding, biological activity, or regulation of growth factors.

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

Régulation de l'expression de PPARγ dans l'arthrose

L’arthrose (OA) est une maladie dégénérative très répondue touchant les articulations. Elle est caractérisée par la destruction progressive du cartilage articulaire, l’inflammation de la membrane synoviale et le remodelage de l’os sous chondral. L’étiologie de cette maladie n’est pas encore bien définie. Plusieurs études ont été menées pour élucider les mécanismes moléculaires et cellulaires impliqués dans le développement de l’OA. Les effets protecteurs du récepteur activé par les proliférateurs de peroxysomes gamma (PPARγ) dans l'OA sont bien documentés. Il a été démontré que PPARγ possède des propriétés anti-inflammatoires et anti-cataboliques. Aussi, plusieurs stimuli ont été impliqués dans la régulation de l’expression de PPARγ dans différents types cellulaires. Cependant, les mécanismes exacts responsables de cette régulation ainsi que le profil de l’expression de ce récepteur au cours de la progression de l’OA ne sont pas bien connus. Dans la première partie de nos travaux, nous avons essayé d’élucider les mécanismes impliqués dans l’altération de l’expression de PPARγ dans cette maladie. Nos résultats ont confirmé l’implication de l’interleukine-1β (IL-1β), une cytokine pro-inflammatoire, dans la réduction de l’expression de PPARγ au niveau des chondrocytes du cartilage articulaire. Cet effet coïncide avec l'induction de l’expression du facteur de transcription à réponse précoce de type 1 (Egr-1). En plus, la diminution de l'expression de PPARγ a été associée au recrutement d'Egr-1 et la réduction concomitante de la liaison de Sp1 au niveau du promoteur de PPARγ. Dans la deuxième partie de nos travaux, nous avons évalué le profil d’expression de ce récepteur dans le cartilage au cours de la progression de cette maladie. Le cochon d’inde avec OA spontanée et le chien avec OA induite par rupture du ligament croisé antérieur (ACLT) deux modèles animaux d’OA ont été utilisés pour suivre l’expression des trois isoformes de PPARs : PPAR alpha (α), PPAR béta (β) et PPAR gamma (γ) ainsi que la prostaglandine D synthase hématopoïétique (H-PGDS) et la prostaglandine D synthase de type lipocaline (L-PGDS) deux enzymes impliquées dans la production de l’agoniste naturel de PPARγ, la 15-Deoxy-delta(12,14)-prostaglandine J(2) (15d-PGJ2). Nos résultats ont démontré des changements dans l’expression de PPARγ et la L-PGDS. En revanche, l’expression de PPARα, PPARβ et H-PGDS est restée stable au fil du temps. La diminution de l’expression de PPARγ dans le cartilage articulaire semble contribuer au développement de l’OA dans les deux modèles animaux. En effet, le traitement des chondrocytes par de siRNA dirigé contre PPARγ a favorisé la production des médiateurs arthrosiques tels que l'oxyde nitrique (NO) et la métalloprotéase matricielle de type 13 (MMP-13), confirmant ainsi le rôle anti-arthrosique de ce récepteur. Contrairement à ce dernier, le niveau d'expression de la L-PGDS a augmenté au cours de la progression de cette maladie. La surexpression de la L-PGDS au niveau des chondrocytes humains a été associée à la diminution de la production de ces médiateurs arthrosiques, suggérant son implication dans un processus de tentative de réparation. En conclusion, l’ensemble de nos résultats suggèrent que la modulation du niveau d’expression de PPARγ, de la L-PGDS et d’Egr-1 au niveau du cartilage articulaire pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et probablement d’autres formes d'arthrite.

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): Histochemical and MMP-1 and -2 and collagen I gene expression analyses

Background: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels.Methods: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies.Results: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group.Conclusion: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

Finasteride treatment alters MMP-2 and-9 gene expression and activity in the rat ventral prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP > AP > DP > VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma. © 2006 International Federation for Cell Biology.

Gene expression changes associated with myocarditis and fibrosis in hearts of mice with chronic chagasic cardiomyopathy

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart.We performed an extensive microarray analysis of hearts from a mouse model of this disease and identified significant alterations in expression of ~12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis (extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide newtherapeutic targets in chronic Chagas disease. © 2010 by the Infectious Diseases Society of America.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

Expression of Protease Activated Receptor-1 in Chronic Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Venomics Profiling of Thamnodynastes strigatus Unveils Matrix Metalloproteinases and Other Novel Proteins Recruited to the Toxin Arsenal of Rear-Fanged Snakes

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus, in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (similar to 47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.