MMP Activity Detection in Zymograms

Bencsik, Péter; Barteková, Monika; Görbe, Anikó; Kiss, Krisztina; Pálóczi, János; Radošinská, Jana; Szűcs, Gergő; Ferdinándy, Péter

doi:10.1007/978-1-4939-7111-4_6

Cited by 25 publications

(32 citation statements)

References 31 publications

(5 reference statements)

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“…In order to investigate MMP-2 and MMP-9 activity [ 54 ] from rat plasma samples, 50 µg of protein/lane was loaded and separated by electrophoresis on a 10% SDS-polyacrylamide gel copolymerized with 2 mg/mL gelatin from porcine skin (G1890, CAS 9000-70-8, Sigma-Aldrich; St. Louis, MO, USA). After electrophoresis, gels were washed in 2.5% TritonX 100 with gentle agitation and then incubated for 20 h at 37 °C in zymography development buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl 2 , 200 mM NaCl).…”

Section: Methodsmentioning

confidence: 99%

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors

Gömöri

Szabados

Kenyeres

et al. 2020

IJMS

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Background: We recently developed novel matrix metalloproteinase-2 (MMP-2) inhibitor small molecules for cardioprotection against ischemia/reperfusion injury and validated their efficacy in ischemia/reperfusion injury in cardiac myocytes. The aim of the present study was to test our lead compounds for cardioprotection in vivo in a rat model of acute myocardial infarction (AMI) in the presence or absence of hypercholesterolemia, one of the major comorbidities affecting cardioprotection. Methods: Normocholesterolemic adult male Wistar rats were subjected to 30 min of coronary occlusion followed by 120 min of reperfusion to induce AMI. MMP inhibitors (MMPI)-1154 and -1260 at 0.3, 1, and 3 µmol/kg, MMPI-1248 at 1, 3, and 10 µmol/kg were administered at the 25th min of ischemia intravenously. In separate groups, hypercholesterolemia was induced by a 12-week diet (2% cholesterol, 0.25% cholic acid), then the rats were subjected to the same AMI protocol and single doses of the MMPIs that showed the most efficacy in normocholesterolemic animals were tested in the hypercholesterolemic animals. Infarct size/area at risk was assessed at the end of reperfusion in all groups by standard Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining, and myocardial microvascular obstruction (MVO) was determined by thioflavine-S staining. Results: MMPI-1154 at 1 µmol/kg, MMPI-1260 at 3 µmol/kg and ischemic preconditioning (IPC) as the positive control reduced infarct size significantly; however, this effect was not seen in hypercholesterolemic animals. MVO in hypercholesterolemic animals decreased by IPC only. Conclusions: This is the first demonstration that MMPI-1154 and MMPI-1260 showed a dose-dependent infarct size reduction in an in vivo rat AMI model; however, single doses that showed the most efficacy in normocholesterolemic animals were abolished by hypercholesterolemia. The further development of these promising cardioprotective MMPIs should be continued with different dose ranges in the study of hypercholesterolemia and other comorbidities.

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Section: Methodsmentioning

confidence: 99%

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors

Gömöri

Szabados

Kenyeres

et al. 2020

IJMS

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“…Gelatin zymography was performed as described previously (Kupai et al, 2010 ; Bencsik et al, 2017 ). MMP-2 was isolated from rat heart homogenates as follows: 50 μg protein/lane were loaded and separated by electrophoresis under non-reducing conditions on an 8% SDS-polyacrylamide gels copolymerized with 2 mg/ml gelatin from porcine skin (Sigma-Aldrich; St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Development of Matrix Metalloproteinase-2 Inhibitors for Cardioprotection

et al. 2018

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The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.

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“…Another histological method for the determination of proteinase activity in tissues is the electrophoretic technique -in situ zymography. Frozen tissue sections are incubated with a fluorescently labeled substrate to determine the location and the quantity of proteins [116].…”

Section: The Level and Activity Of Proteinasesmentioning

confidence: 99%

Extracellular Matrix Markers and Methods for Their Study (Review)

Tush¹,

Eliseeva²,

Khaletskaya³

et al. 2019

Sovrem Tehnol Med

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The extracellular matrix (ECM) is a complex meshwork consisting mainly of proteins and carbohydrates; it is currently viewed as a key factor of tissue organization and homeostasis. In each organ, the composition of ECM is different: it includes a variety of fibrillar components, such as collagens, fibronectin, and elastin, as well as non-fibrillar molecules: proteoglycans, hyaluronan, glycoproteins, and matrix proteins. ECM is an active tissue, where the de novo syntheses of structural components are constantly taking place. In parallel, ECM components undergo degradation catalyzed by a number of enzymes including matrix metalloproteinases. The synthesis and degradation of ECM components are controlled by mediators and cytokines, metabolic, epigenetic, and environmental factors. Currently, a large amount of evidence indicates that modifications (remodeling) of ECM play an important role in the pathogenesis of clinical conditions. This may explain the increasing interest in the markers of ECM remodeling both in health and disease. In recent years, many of the ECM markers were considered targets for diagnosing, predicting, and treating diseases. In this review, we discuss some of the currently known ECM markers and methods used for their determination.

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MMP Activity Detection in Zymograms

Cited by 25 publications

References 31 publications

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors

Development of Matrix Metalloproteinase-2 Inhibitors for Cardioprotection

Extracellular Matrix Markers and Methods for Their Study (Review)

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