Search

Matrix metalloproteinases (MMPs) are enzymes that present a metallic element in their structure. These enzymes are ubiquitously distributed and function as extracellular matrix (ECM) remodelers. MMPs play a broad role in cardiovascular biology regulating processes such as cell adhesion and function, cellular communication and differentiation, integration of mechanical force and force transmission, tissue remodeling, modulation of damaged-tissue structural integrity, cellular survival or apoptosis and regulation of inflammation-related cytokines and growth factors. MMPs inhibition and downregulation are correlated with minimization of cardiac damage, i.e., Chinese herbal medicine has shown to stabilize abdominal aorta aneurysm due to its antiinflammatory, antioxidant and MMP-2 and 9 inhibitory properties. Thus phyto-derived products rise as promising sources for novel therapies focusing on MMPs inhibition and downregulation to treat or prevent cardiovascular disorders.

A ubaia (Eugenia patrisii Vahl.) é uma frutífera nativa da Amazônia cujos frutos são consumidos in natura e na forma de suco. Atualmente a produção desta espécie ainda está restrita ao extrativismo, existindo poucas informações sobre o cultivo e manejo. Neste sentido esta pesquisa avaliou o crescimento vegetativo e a produtividade de frutos de E. patrisii em uma área de cultivo em diferentes condições de adubação. O experimento foi conduzido na Área de Pesquisa Experimental do Instituto Federal de Ciências e Tecnologia do Pará, município de Marabá, Pará, Brasil. As mudas foram produzidas a partir de sementes e cultivadas em condição de sequeiro. Utilizou-se o delineamento em blocos casualizado com três tratamentos: (Argissolo Vermelho-Amarelo + N, P, K); (Argissolo Vermelho-Amarelo + Esterco bovino); (Argissolo Vermelho-Amarelo). A unidade experimental foi composta por 6 plantas, com dez repetições em cinco blocos. Foram avaliados padrões de desenvolvimento vegetal: altura total; diâmetro do colmo; massa seca da parte aérea; massa seca do sistema radicular; massa seca total; relação entre a altura da parte aérea e o diâmetro do colo; relação entre a massa seca da parte aérea e a massa seca do sistema radicular, e Índice de Qualidade de Dickson. Também foram avaliados os parâmetros de produção de frutos: número de frutos; massa média dos frutos e produção de frutos e fenologia. As mudas de ubaia apresentaram desenvolvimento e produção de frutos em todas as condições experimentais com destaque para as cultivadas em tratamento orgânico (Argissolo Vermelho-Amarelo + Esterco Bovino) que apresentaram melhor desenvolvimento e produtividade.

Atherogenic events promote changes in vessel walls, with alteration of the redox state, and increased activity of matrix metalloproteinases (MMPs). Thus, this study aims to evaluate aortic remodeling, MMP activity, and reactive oxygen species (ROS) levels after treatment with doxycycline in ApoE-⁄- and ovariectomized mice (OVX). Female ApoE-⁄--knockout mice (5 weeks) were submitted to ovariectomy surgery to induce experimental menopause. They then received chow enriched with 1% cholesterol to induce hypercholesterolemia. The animals were divided into two experimental groups: ApoE-⁄-/OVX vehicle and ApoE-⁄-/OVX doxycycline (30 mg/kg) administered by gavage once a day for 28 days (15th to the 18th week of life). Blood samples were collected to measure total cholesterol and fractions. The aorta was used for morphometry and to measure the activity and expression of MMP-2 and ROS levels. The ApoE-⁄-/OVX doxycycline group showed no change in total and fraction cholesterol levels. However, there was a reduction in ROS levels, MMP-2 expression, and activity that correlated with a decrease in atherosclerotic lesions relative to the ApoE-⁄-/OVX vehicle (p > 0.05). Therefore, we conclude that doxycycline in ApoE-⁄-/OVX animals promotes a reduction in atherosclerotic lesions by reducing ROS and MMP-2 activity and expression.

Atherogenic events promote changes in vessel walls, with alteration of the redox state, and increased activity of matrix metalloproteinases (MMPs). Thus, this study aims to evaluate aortic remodeling, MMP activity, and reactive oxygen species (ROS) levels after treatment with doxycycline in

Various pathophysiological mechanisms have been implicated in hypertension, but those resulting in vascular dysfunction and remodeling are critical and may help to identify critical pharmacological targets. This mini-review article focuses on central mechanisms contributing to the vascular dysfunction and remodeling of hypertension, increased oxidative stress and impaired nitric oxide (NO) bioavailability, which enhance vascular matrix metalloproteinase (MMP) activity. The relationship between NO, MMP and oxidative stress culminating in the vascular alterations of hypertension is examined. While the alterations of hypertension are not fully attributable to these pathophysiological mechanisms, there is strong evidence that such mechanisms play critical roles in increasing vascular MMP expression and activity, thus resulting in abnormal degradation of extracellular matrix components, receptors, peptides, and intracellular proteins involved in the regulation of vascular function and structure. Imbalanced vascular MMP activity promotes vasoconstriction and impairs vasodilation, stimulating vascular smooth muscle cells (VSMC) to switch from contractile to synthetic phenotypes, thus facilitating cell growth or migration, which is associated with the deposition of extracellular matrix components. Finally, the protective effects of MMP inhibitors, antioxidants and drugs that enhance vascular NO activity are briefly discussed. Newly emerging therapies that address these essential mechanisms may offer significant advantages to prevent vascular remodeling in hypertensive patients.

A poluicao ambiental ocasionada por metais toxicos tem aumentado com o avanco da atividade industrial e agricola. Dentre as tecnicas de tratamento de ambientes contaminados, destaca-se a fitorremediacao: por ser um metodo eficiente, barato e inofensivo ao meio ambiente ja que utiliza plantas para realizar o tratamento. Estudos realizados com a Montrichardia linifera, uma macrofita aquatica encontrada vastamente as margens dos rios amazonicos, mostram que a planta absorve altas concentracoes de manganes, o que pode ser um indicativo de bioacumulacao deste metal por parte da planta. Desta forma, o objetivo deste estudo foi avaliar o potencial bioacumulador e fitorremediador da parte aerea de M. linifera comparando os teores de metais encontrados na planta com os encontrados no solo. As amostras de folha, peciolo, caule e solo foram coletadas no aningal do campus pesquisa do Museu Paraense Emilio Goeldi e determinados os teores de Ca, Mg, Cu, Fe, Zn, Mn, Pb, Cd, Co e Ni do material seco em espectrometro de absorcao atomica de chama. A M. linifera apresentou alta bioacumulacao de quase todos os metais analisados, principalmente manganes nas folhas (149,11 µg g-1) e zinco no peciolo (10,36 mg g-1) com cerca de 16,5 e 10,7 vezes, respectivamente, a concentracao presente no solo. Os resultados mostram que planta acumula grandes quantidades de manganes, zinco, cadmio e cobre em sua parte aerea, com teores bem superiores ao disponivel no solo, possuindo assim grande potencial para aplicacao na fitorremediacao de ambientes contaminados por esses metais.

Osteoporosis and cardiovascular diseases are common causes of morbidity and mortality worldwide, especially in people aged over 60 years. Osteoporosis is characterized by low bone mineral density, which deteriorates the microarchitecture of bones and increases the risk of bone fractures. Other pathologies also constitute risk factors for the development of osteoporosis, mainly cardiovascular diseases. In fact, a growing number of reports have shown a positive correlation between cardiovascular diseases and low bone mineral density. MMPs are proteases that participate in the organized degradation of the extracellular matrix (ECM) and which play essential physiological roles, such as cardiovascular and bone tissue remodeling. Overexpression of MMPs underlies pathological processes like osteoporosis and cardiovascular diseases. MMP-1, -2, -9, -13, and -14 are expressed in bone tissue and are key players in the digestion of bone matrix by osteoblasts. Considering this relationship between osteometabolic and cardiovascular pathologies and MMPs, this review focuses on the involvement of MMPs in osteoporosis and on their participation in cardiovascular diseases; it also deals with the positive correlation between osteoporosis and cardiovascular diseases. Although there are many drugs to treat osteoporosis, controversies exist. Here, we will describe these controversies and will discuss how inhibition of MMPs could be an alternative strategy to or an adjuvant therapy in the current treatment of osteoporosis.

This work was supported by the Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq), This study was financed in part by the Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior ? Brasil (CAPES) Finance code 001, PROPESP-UFPA and Instituto Nacional de Biologia Estrutural e Bioimagem-INBEB [CNPq ? Grant number 465395/2014 ]. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Leishmaniose Prof. Dr. Ralph Lainson. Ananindeua, PA, Brasil / Federal University of Para. Institute of Biological Sciences. Laboratory of Structural Biology. Bel?m, PA, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Structural Biology. Bel?m, PA, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Structural Biology. Bel?m, PA, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Microscopia Eletr?nica. Ananindeua, PA, Brasil. Federal University of Para. Institute of Biological Sciences. Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/? 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/? 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.

Glioblastoma, which is highly invasive and has a poor patient prognosis, is the most common type of brain tumor. Flavonoids have known antiproliferative and antineoplastic effects, such as apoptosis induction and tumor growth inhibition. We investigated the effects of treatment with three flavonoids (BAS-1, BAS-4, and BAS-6) isolated from the Amazon plant Brosimum acutifolium on the proliferation and migration of the C6 glioma cell line. Cytotoxicity was evaluated by MTT assay, and morphological changes were evaluated by phase-contrast microscopy and by transmission electron microscopy. Apoptosis was determined using Annexin V-FITC-propidium iodide (PI) staining. A hemolysis assay was used to evaluate plasma membrane injury. Antiproliferative effects were assessed by wound migration and colony formation assays. Mitochondrial transmembrane potential (ΔΨm) was determined using JC-1 dye and flow cytometry. To identify the flavonoid targets, western blotting was performed. BAS-1 and BAS-4 reduced C6 cell proliferation in a dose-dependent manner. BAS-6 showed no effect. Due to its high toxicity toward primary glial cells and its high hemolytic index, BAS-1 was not used in the remaining experiments. BAS-4 treatment did not induce cytotoxicity in primary glial cells; however, in glioma cells, it suppressed migration and invasion and led to apoptosis through mitochondrial damage, ΔΨm loss, cell cycle arrest, and reduced AKT phosphorylation, which is a component of the main cell survival pathway. We conclude that BAS-4 showed potential activity against glioma by inducing apoptosis mediated by ΔΨm loss and AKT pathway disruption, and future studies should further evaluate BAS-4 as a promising antineoplastic agent against glioblastoma.

Aims Hypertension underlies endothelial dysfunction, and activation of vasorelaxation signaling with low dependence on nitric oxide (NO) represents a good alternative for vascular modulation. C-type natriuretic peptide (CNP) causes relaxation by increasing cyclic guanosine 3′,5′-monophosphate (cGMP) or Gi-protein activation through its natriuretic peptide receptor-B or -C, respectively. We have hypothesized that CNP could exerts its effects and could overcome endothelial dysfunction in two kidney-one clip (2K-1C) hypertensive rat aorta. Here, we investigate the intracellular signaling involved in CNP effects in hypertension. Materials and methods The 2K-1C hypertension was induced in male Wistar rats (200 g). CNP-induced vascular relaxation and cGMP production were investigated in rat thoracic aortas. The natriuretic peptide receptor-B and -C localization was evaluated by immunofluorescence. Calcium mobilization was assessed in endothelial cells from rat aortas. Key findings CNP induced similar relaxation in normotensive and 2K-1C hypertensive rat aortas, which increased after endothelium removal. CNP-induced relaxation involved natriuretic peptide receptor-B and -C activation in 2K-1C rats. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) counter-regulated CNP-particulate GC (pGC) activation in aortas. CNP reduced endothelial calcium and increased cGMP production, which was lower in 2K-1C. CNP-induced cGMP-dependent protein kinase (PKG) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activation was impaired in 2K-1C rat aorta. Significance Our results indicated CNP triggered relaxation through its natriuretic peptide receptor-B and -C in 2K-1C rat aortas, and that CNP-induced relaxation overcomes endothelial dysfunction in hypertension. In addition, NOS and sGC activities counter-regulate CNP-pGC activation to induce vascular relaxation.

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases., Graphical abstract fx1, Highlights • MMP-2 is activated by reactive oxygen species and promotes cardiovascular diseases. • We show here that MMP-2 is upstream of reactive oxygen species formation. • This effect involves epidermal growth factor receptor transactivation. • MMP-2 impairs redox balance and contributes to vascular contraction.

This MiniReview describes the essential biochemical and molecular aspects of matrix metalloproteinases (MMPs) and briefly discusses how they engage in different diseases, with particular emphasis on cardiovascular diseases. There is compelling scientific evidence that many MMPs, especially MMP-2, play important roles in the development of cardiovascular diseases; inhibition of these enzymes is beneficial to many cardiovascular conditions, sometimes precluding or postponing end-organ damage and fatal outcomes. Conducting comprehensive discussions and further studies on how MMPs participate in cardiovascular diseases is important, because inhibition of these enzymes may be an alternative or an adjuvant for current cardiovascular disease therapy.

This study aimed to investigate the effects of Coriandrum sativum aqueous extract (CSAE) on the rat progeny of mothers exposed to methylmercury (MeHg). The presence of bioactive compounds and CSAE's antioxidant capacity been evaluated, and the offspring were assessed for their total mercury levels, motor behavioral parameters and oxidative stress in the cerebellum. The analysis of the bioactive compounds revealed significant amounts of polyphenols, flavonoids, and anthocyanins, as well as a variety of minerals. A DPPH test showed the CSAE had important antioxidant activity. The MeHg + CSAE group performed significantly better spontaneous locomotor activity, palmar grip strength, balance, and motor coordination in behavioral tests compared the MeHg group, as well as in the parameters of oxidative stress, with similar results to those of the control group. The MeHg + CSAE group also had significantly reduced mercury levels in comparison to the MeHg group. Based on the behavioral tests, which detected large locomotor, balance, and coordination improvements, as well as a reduction in oxidative stress, we conclude that CSAE had positive functional results in the offspring of rats exposed to MeHg.

There is an imbalance between contractile and relaxing endothelial factors in hypertension which induces vascular dysfunction. It involves cyclooxygenase (COX) due to production of vasoconstrictor agonists such as thromboxane A2 (TXA2). In this work we used the NO-NSAID vasodilator compound NCX2121 that has nitric oxide (NO) in its structure and the non-selective COX inhibitor indomethacin. This class of pro-drug has been demonstrated hypotensive effect in animal models of hypertension without alteration in the arterial pressure of normotensive rats. It has been pointed a direct vasodilator effect of NO-NSAIDS which mechanism remains unclear. This study aimed to evaluate the cellular mechanisms involved in the NCX2121-induced relaxation in renal hypertensive two kidney-one clip (2K-1C) rat aorta with intact endothelium and denuded aortas. Our data demonstrated that NO was directly released from the compound NCX2121 in the cytosol of the vascular smooth muscle cells. The relaxation induced by NCX2121 was similar in 2K-1C and normotensive two kidney (2K). It was impaired by the endothelium removal and NO-Synthase (NOS) inhibition in both rat aorta groups. The inhibition of the main NO target, soluble guanylyl-cyclase (sGC) with ODQ, abolished NCX2121-induced relaxation in denuded aorta. The expression of COX-1 and COX-2 as well the produced stable analog of Thromboxane A2 (TXA2) were higher in 2K-1C than in 2K. NCX2121 decreased the augmented production of TXA2 in 2K-1C rat aortas. In intact-endothelium aorta, ODQ impaired the relaxation induced by NCX2121 in 2K but it had no effect in 2K-1C rat aorta. Taken together, our results show that NCX2121-induced relaxation is due to the direct NO release inside the vascular smooth muscle cells and sGC activation in denuded aortas, whereas in intact endothelium aortas, NCX2121-induced relaxation could be due to eNOS activation and inhibition of prostanoids production. In addition, relaxation-induced by NCX2121 is not greater in normotensive than in hypertensive rat aortas because COX activity is higher in 2K-1C than in 2K rat aortas.

Background: An increase in MMP-2 levels is reported in heart failure (HF). However the role of MMP-2 in the pathogenesis of HF remains unclear. The aim of the present study was to determine the effect of increased circulating levels of MMP-2 on heart morphology and function. Methods and Results: Purified MMP-2 catalytic domain fused to GFP (catMMP-2/GFP) or saline (control) was injected into 11-wk-old male C57BL/6 mice for four weeks. The fluorescent active protein was tracked in vivo and homed in the heart. Cardiomyocyte diameter not changed between groups (catMMP-2/GFP: 11.37 ± 0.25 μm, n=7; Control: 11.38 ± 0.13 μm, n=7; P =0.97). On the other hand, fibrosis increased in the hearts of catMMP-2/GFP mice (0.82 ± 0.05% area/field, n=7 vs Control: 0.58 ± 0.02% area/field, n=7; P< 0.05). Apoptotic stained nuclei in the left ventricle (LV) of catMMP-2/GFP injected-mice amounted to 7.24%, n=4, PP P P P P Conclusions: Circulating active MMP-2 homing to the heart interstitium degrades N-cadherin and induces TGF-β1 overexpression, leading to apoptosis, and fibrosis. This mechanism may account for heart function loss when plasma levels of MMP-2 increase.

Aims: To evaluate the role of nitric oxide, reactive oxygen species, and natriuretic peptide receptor-B activation in C-type natriuretic peptide-anti-contractile effect on Phenylephrine-induced contraction in aorta isolated from septic rats. Methods and Results: Cecal ligation and puncture surgery was used to induce sepsis in male rats. Vascular reactivity was conducted in rat aorta and resistance mesenteric artery. Measurement of survival rate, mean arterial pressure, plasma nitric oxide, specific protein expression and localization were evaluated. Septic rats had a survival rate about 37% at 4h after the surgery, and these rats presented hypotension compared to control-operated (Sham) rats. Phenylephrine-induced contraction was decreased in sepsis. C-type natriuretic peptide induced anti-contractile effect in aortas. Plasma nitric oxide was increased in sepsis. Nitric oxide-synthase but not natriuretic peptide receptor-B expression was increased in septic rat aortas. C-type natriuretic peptide-anti-contractile effect was dependent on nitric oxide-synthase, reactive oxygen species and natriuretic peptide receptor-B activation. Natriuretic peptide receptor-C, protein kinase-Cα mRNA and basal nicotinamide adenine dinucleotide phosphate-dependent reactive oxygen species production were lower in septic rats. Phenylephrine and C-type natriuretic peptide enhanced reactive oxygen species production. However, stimulated reactive oxygen species production was low in sepsis. Conclusion: C-type natriuretic peptide induced anti-contractile effect on Phenylephrine contraction in aortas from Sham and septic rats that was dependent on nitric oxide-synthase, reactive oxygen species and natriuretic peptide receptor-B activation. Keywords: C-type natriuretic peptide – Sepsis – Natriuretic peptide receptor B – Reactive oxygen species – Nitric oxide- Phenylephrine-induced contraction

Imbalanced matrix metalloproteinase (MMP)-2 activity may result of enhanced reactive oxygen species (ROS) formation and promotes cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the NADPH oxidase inhibitor apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

Matrix Metalloproteinases (MMPs) participate in many physiological and pathological processes. One major limitation to a better understanding of the role MMPs play in these processes is the lack of well-characterized chimeric proteins and characterization of their fluorescence. The specialized literature has reported on few constructs bearing MMPs fused to the sequence of the green fluorescent protein (GFP), but none of the described constructs have been intended for expression in bacteria or for purification and use in vivo. This work has tested a recombinant reporter protein containing the MMP-2 catalytic domain fused to GFP in terms of purification efficiency, degradation of substrates in solution and in zymograms, kinetic activity, GFP fluorescence, and GFP fluorescence in whole animals after injection of the purified and lyophilized fluorescent protein. This work has also characterized rhMMP-2 (recombinant human MMP-2) and inactive clones and used them as negative controls in experiments employing catMMP-2/GFP and rhMMP-2. To our knowledge, this is the first study that has fully characterized a chimeric protein with the MMP-2 catalytic domain fused to GFP, that has efficiently purified such protein from bacteria in a single-step, and that has obtained an adequate chimeric protein for injection in animals and tracking of MMP-2 fate and activity in vivo.

Hyperglycemia increases the generation of reactive oxygen species and affects systems that regulate the vascular tone including renin-angiotensin system. Stress could exacerbate intracellular oxidative stress during Diabetes upon the activation of angiotensin AT1/NADPH oxidase pathway, which contributes to the development of diabetic cardiovascular complications. For this study, type-I Diabetes was induced in Wistar rats by intraperitoneal injection of streptozotocin. 28 days after streptozotocin injection, the animals underwent to acute restraint stress for 3 h. Cumulative concentration-response curves for angiotensin II were obtained in carotid rings pre-treated or not with Nox or cyclooxygenase inhibitors. Nox1 or Nox4 expression and activity were assessed by Western blotting and lucigenin chemiluminescence, respectively. The role of Nox1 and Nox4 on reactive oxygen species generation was evaluated by flow cytometry and Amplex Red assays. Cyclooxygenases expression was assessed by real-time polymerase chain reaction. The contractile response evoked by angiotensin II was increased in diabetic rat carotid. Acute restraint stress increased this response in this vessel by mechanisms mediated by Nox4, whose local expression and activity in generating hydrogen peroxide are increased. The contractile hyperreactivity to angiotensin II in stressed diabetic rat carotid is also mediated by metabolites derived from cyclooxygenase-2, whose local expression is increased. Taken together, our findings suggest that acute restraint stress exacerbates the contractile hyperreactivity to angiotensin II in diabetic rat carotid by enhancing Nox4-driven generation of hydrogen peroxide, which evokes contractile tone by cyclooxygenases-dependent mechanisms. Finally, these findings highlight the harmful role played by acute stress in modulating diabetic vascular complications.

AT1 antagonists effectively prevent atherosclerosis since AT1 upregulation and angiotensin II-induced proinflammatory actions are critical to atherogenesis. Despite the classic mechanisms underlying the vasoprotective and atheroprotective actions of AT1 antagonists, the cross-talk between angiotensin-converting enzyme-angiotensin II-AT1 and angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axes suggests other mechanisms beyond AT1 blockage in such effects. For instance, angiotensin-converting enzyme 2 activity is inhibited by reactive oxygen species derived from AT1-mediated proinflammatory signaling. Since angiotensin-(1-7) promotes antiatherogenic effects, we hypothesized that the vasoprotective and atheroprotective effects of AT1 antagonists could result from their inhibitory effects on the AT1-mediated negative modulation of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality. Interestingly, our results showed that early atherosclerosis triggered in thoracic aorta from high cholesterol fed-Apolipoprotein E-deficient mice impairs angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality by a proinflammatory-redox AT1-mediated pathway. In such mechanism, AT1 activation leads to the aortic release of tumor necrosis factor-α, which stimulates NAD(P)H oxidase/Nox1-driven generation of superoxide and hydrogen peroxide. While hydrogen peroxide inhibits angiotensin-converting enzyme 2 activity, superoxide impairs MAS functionality. Candesartan treatment restored the functionality of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis by inhibiting the proinflammatory-redox AT1-mediated mechanism. Candesartan also promoted vasoprotective and atheroprotective effects that were mediated by MAS since A779 (MAS antagonist) co-treatment inhibited them. The role of MAS receptors as the final mediators of the vasoprotective and atheroprotective effects of candesartan was supported by the vascular actions of angiotensin-(1-7) upon the recovery of the functionality of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis.

The matrix metalloproteinases (MMPs) are known for quite some time to play essential roles in this remodeling process of arteries found in hypertension, as will be discussed here. More recently, evidence indicates that some of them may not only influence hypertension due to their effect in remodeling but possibly by direct mechanisms such as the proteolytic change of vasoactive mediators. The most studied MMP in animal models of hypertension is MMP-2, and many such studies are described here. After a brief description of MMPs, we discuss data on MMPs in hypertension in three sections: findings on arteries, findings on the heart, and on the final section we then briefly discuss some other issues that may indicate that MMPs are related to hypertension in other yet unexplored ways. In this review we also highlight data showing the protective effects of MMP inhibition in arteries, heart, and kidney. MMP inhibition appears to be a relevant target for newer pharmacological intervention in hypertension. Doxycycline is the MMP inhibitor most widely used in cell cultures, animal studies on hypertension, and is a drug approved for commercial use for periodontal disease in the United States. The refinement of MMP assays and the specific delivery of different MMPs may highlight the specific role of each MMP in the different types of hypertension, enabling the use of a rational approach for a more specific inhibition of them in hypertension, so that target organs are more protected.

Endothelial nitric oxide negatively modulates Phenylephrine-induced contraction in Matrix metalloproteinase 2 (MMP-2)-filled rabbit aortic rings The present study aimed to evaluate the vascular function of aortic rings after intraluminal injection of matrix metalloproteinase 2 (MMP-2) and contribution of endothelial nitric oxide (NO) in this response. METHODS: thoracic aorta isolated from New Zealand rabbits (3.0 Kg) was isolated and were performed in situ gelatinolytic assay (intraluminal exposure with Krebs solution or MMP-2 1.2 μg/mL, at 37° C for 30 minutes, with measure of fluorescence intensity - FI) or vascular reactivity. Cumulative concentration-effect curves for phenylephrine (PE, 0.1 nM - 10 μM/L) were performed in endothelium-intact (E+) or denuded (E-) aortic rings, in the absence or presence of L-NAME (non-selective inhibitor of NOS, 100 μM) or Hydroxycobalamine (scavenger of intracellular NO), 30 min before the PE curves on E+ rings. The vascular contraction was normalized by grams of tension by dry tissue (g/g) and were analyzed the agonist maximum effect (Emax) and potency (pD2). Data represent mean ± S.E.M and the results were statistically analyzed using Student t test, one-way or two-way ANOVA with Bonferroni pos-hoc (p Results: In situ gelatinolytic activity was increased in the vessels filled with MMP-2 (19.19+0.93U,n=5) compared to vehicle (10.75+0.45U,n=5). In the vascular reactivity, E- aortic rings filled with MMP-2 increased Emax of PE (1893,28+122,40g/g,n=9) versus vehicle E- (1485.73+76.82g/g,n=9), but no differences were observed on Emax of E+ aortic rings (vehicle: 1416.13+120.87g/g,n=7; MMP-2: 1893.28+122.40g/g,n=9). The presence of L-NAME induced an increase in Emax of E+ aortic rings filled with vehicle (1817.60+76.37g/g,n=7) or MMP-2 (2340.50+130.43g/g,n=5). Hydroxycobalamine also increased the Emax of E+ aortic rings with vehicle (1889,81+129,17,n=6) or MMP-2 (2480,12+192,57,n=5). No difference of pD2 value was observed in this study. Conclusion: Data suggest this protease cross the aorta layers from the lumen. An increased vasoconstriction in E- vessels suggests a vascular smooth muscle activity of MMP-2. In the presence of L-NAME and Hydroxycobalamine, vessels filled with MMP-2 shows a greater maximum effect suggesting that NO exerts a negative modulation of the PE-induced vasoconstriction on MMP-2 aortic rings.