Aline Azevedo

Matrix metalloproteinases are involved in cardiovascular diseases.

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.

C-Type Natriuretic Peptide Induces Anti-contractile Effect Dependent on Nitric Oxide, Oxidative Stress, and NPR-B Activation in Sepsis

Aims: To evaluate the role of nitric oxide, reactive oxygen species (ROS), 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 (CLP) surgery was used to induce sepsis in male rats. Vascular reactivity was conducted in rat aorta and resistance mesenteric artery (RMA). Measurement of survival rate, mean arterial pressure (MAP), plasma nitric oxide, specific protein expression, and localization were evaluated. Septic rats had a survival rate about 37% at 4 h 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 (CNP) 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, ROS, and natriuretic peptide receptor-B activation. Natriuretic peptide receptor-C, protein kinase-Cα mRNA, and basal nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were lower in septic rats. Phenylephrine and CNP enhanced ROS production. However, stimulated ROS production was low in sepsis. Conclusion: CNP induced anti-contractile effect on Phenylephrine contraction in aortas from Sham and septic rats that was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

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.

MMPs are involved in osteoporosis and are correlated with cardiovascular diseases

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.

Matrix metalloproteinase 2 fused to GFP, expressed in E. coli, successfully tracked MMP-2 distribution in vivo.

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.

Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels

Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.

The use of a DNA stabilizer in human dental tissues stored under different temperature conditions and time intervals

Objective The present study evaluated the use of a reagent to stabilize the DNA extracted from human dental tissues stored under different temperature conditions and time intervals. Material and Methods A total of 161 teeth were divided into two distinct groups: intact teeth and isolated dental pulp tissue. The samples were stored with or without the product at different time intervals and temperature. After storage, DNA extraction and genomic DNA quantification were performed using real-time PCR; the fragments of the 32 samples that represented each possible condition were analyzed to find the four pre-selected markers in STR analysis. Results The results of the quantification showed values ranging from 0.01 to 10,246.88 ng/μL of DNA. The statistical difference in the quantity of DNA was observed when the factors related to the time and temperature of storage were analyzed. In relation to the use of the specific reagent, its use was relevant in the group of intact teeth when they were at room temperature for 30 and 180 days. The analysis of the fragments in the 32 selected samples was possible irrespective of the amount of DNA, confirming that the STR analysis using an automated method yields good results. Conclusions The use of a specific reagent showed a significant difference in stabilizing DNA in samples of intact human teeth stored at room temperature for 30 and 180 days, while the results showed no justification for using the product under the other conditions tested.