Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ)

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMD_J) model, a suitable animal model for Duchenne muscular dystrophy.</p> <p>Methods</p> <p>We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMD_Jdogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and <it>in situ </it>zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.</p> <p>Results</p> <p>In CXMD_Jskeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMD_Jmuscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMD_Jmuscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMD_Jdid not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMD_Jdogs compared to the levels observed in normal controls.</p> <p>Conclusion</p> <p>MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.</p

PI16 is a shear stress and inflammation-regulated inhibitor of MMP2

Raised endothelial shear stress is protective against atherosclerosis but such protection may be lost at sites of inflammation. We found that four splice variants of the peptidase inhibitor 16 (PI16) mRNA are among the most highly shear stress regulated transcripts in human coronary artery endothelial cells (HCAECs), in vitro but that expression is reduced by inflammatory mediators TNFα and IL-1β. Immunohistochemistry demonstrated that PI16 is expressed in human coronary endothelium and in a subset of neointimal cells and medial smooth muscle cells. Adenovirus-mediated PI16 overexpression inhibits HCAEC migration and secreted matrix metalloproteinase (MMP) activity. Moreover, PI16 inhibits MMP2 in part by binding an exposed peptide loop above the active site. Our results imply that, at high endothelial shear stress, PI16 contributes to inhibition of protease activity; protection that can be reversed during inflammation

A novel peak detection approach with chemical noise removal using short-time FFT for prOTOF MS data.

Peak detection is a pivotal first step in biomarker discovery from MS data and can significantly influence the results of downstream data analysis steps. We developed a novel automatic peak detection method for prOTOF MS data, which does not require a priori knowledge of protein masses. Random noise is removed by an undecimated wavelet transform and chemical noise is attenuated by an adaptive short-time discrete Fourier transform. Isotopic peaks corresponding to a single protein are combined by extracting an envelope over them. Depending on the S/N, the desired peaks in each individual spectrum are detected and those with the highest intensity among their peak clusters are recorded. The common peaks among all the spectra are identified by choosing an appropriate cut-off threshold in the complete linkage hierarchical clustering. To remove the 1 Da shifting of the peaks, the peak corresponding to the same protein is determined as the detected peak with the largest number among its neighborhood. We validated this method using a data set of serial peptide and protein calibration standards. Compared with MoverZ program, our new method detects more peaks and significantly enhances S/N of the peak after the chemical noise removal. We then successfully applied this method to a data set from prOTOF MS spectra of albumin and albumin-bound proteins from serum samples of 59 patients with carotid artery disease compared to vascular disease-free patients to detect peaks with S/N> or =2. Our method is easily implemented and is highly effective to define peaks that will be used for disease classification or to highlight potential biomarkers.link_to_OA_fulltex