Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids

ObjectivesTo explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis.MethodsLatent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines.ResultsAll MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort.ConclusionSynovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

Parameters of the gelatinase B activity in the integument epithelium and the lamina propria of the gastric mucosa in experimental acetate ulcer in rats

Objective: to determine the specifi c number and expression intensity of gelatinase B-positive cells in the gastric surface epithelium and lamina propria (cell component) in the area of the ulcerous defect in rats with experimental acetate ulcer of the stomach.Materials and methods: 18 white Wistar rats were divided into three groups: initial (intact), experimental (modeling of an acetate ulcer) and control (falsely operated) in the experiment. Tissues were obtained from the ulceration zone or the presumed ulceration zone (in the initial and control groups) to asses morphological changes and also indicators, characterizing the activity of gelatinase B 7 days after the simulation of the experiment.Results and Discussion. It was revealed some increase in the specifi c number of gelatinase B-positive cells against the background of moderate accumulation of lymphocytes in the integument epithelium of the gastric mucosa. A signifi cant increase in the specifi c number of gelatinase B-positive cells at the maximum intensity of its expression and a moderate accumulation of eff ector cells of tissue alterations was determined in the cellular component of the lamina propria of the gastric mucosa at the same time.

Anti-inflammatory Effects of S. cumini Seed Extract on Gelatinase-B (MMP-9) Regulation against Hyperglycemic Cardiomyocyte Stress

Black berry (Syzygium cumini) fruit is useful in curing diabetic complications; however, its role in diabetes-induced cardiomyopathy is not yet known. In this study, we investigated the regulation of gelatinase-B (MMP-9) by S. cumini methanol seed extract (MSE) in diabetic cardiomyopathy using real-time PCR, RT-PCR, immunocytochemistry, gel diffusion assay, and substrate zymography. The regulatory effects of MSE on NF-κB, TNF-α, and IL-6 were also examined. Identification and estimation of polyphenol constituents present in S. cumini extract were carried out using reverse-phase HPLC. Further, in silico docking studies of identified polyphenols with gelatinase-B were performed to elucidate molecular level interaction in the active site of gelatinase-B. Docking studies showed strong interaction of S. cumini polyphenols with gelatinase-B. Our findings indicate that MSE significantly suppresses gelatinase-B expression and activity in high-glucose- (HG-) stimulated cardiomyopathy. Further, HG-induced activation of NF-κB, TNF-α, and IL-6 was also remarkably reduced by MSE. Our results suggest that S. cumini MSE may be useful as an effective functional food and dietary supplement to regulate HG-induced cardiac stress through gelatinase.

Magnesium-containing composition influencing morphological parameters and gelatinase B activity in endometrial tissues in experimental chronic endometrial inflammation

Aim: to assess an effect of medicinal magnesium-containing composition on gelatinase B expression intensity and morphological parameters of chronic experimental endometrial inflammation.Materials and Methods. There were conducted experiments with 60 sexually mature female Wistar rats to determine an effect of medicinal magnesium-containing composition (contains magnesium chloride – a natural polymineral Bischofite) on gelatinase B activity in endometrial tissues (counting number of gelatinase-B-positive cells and intensity of gelatinase B expression), morphological parameters of cell infiltration, as well as amount of magnesium in red blood cell mass collected from the inferior vena cava and subclavian vein. All such parameters were assessed in experimental animals from 4 groups: group 1 – animals in baseline state, group 2 – control, group 3 – experimental chronic endometritis (CE), group 4 – experimental CE after treatment with medicinal magnesium-containing composition. Results. It was found that use of medicinal magnesium-containing composition increased amount of erythrocyte magnesium up to the baseline level and increased both number and expression intensity of stromal gelatinase B-positive cells. In addition, magnesium level in erythrocyte mass from the inferior vena cava was increased and accompanied with restored eosinophil-plasmacyte as well as significantly elevated macrophage-lymphocyte infiltration in endometrial tissues compared to CE animals lacking therapy. Conclusion. The results of this study allow us to conclude about importance of gelatinase B in pathogenesis of experimental chronic endometrial inflammation as well as an opportunity of regulating gelatinase B activity by applying medicinal magnesium containing drug in pathogenetic therapy of experimental CE.