Musculoskeletal disorders include rheumatoid arthritis, osteoarthritis, sarcopenia, injury, stiffness, and bone loss. The prevalence of these conditions is frequent among elderly populations with significant mobility and mortality rates. This may lead to extreme discomfort and detrimental effect on the patient’s health and socioeconomic situation. Muscles, ligaments, tendons, and soft tissue are vital for body function and movement. Matrix metalloproteinases (MMPs) are regulatory proteases involved in synthesizing, degrading, and remodeling extracellular matrix (ECM) components. By modulating ECM reconstruction, cellular migration, and differentiation, MMPs preserve myofiber integrity and homeostasis. In this review, the role of MMPs in skeletal muscle function, muscle injury and repair, skeletal muscle inflammation, and muscular dystrophy and future approaches for MMP-based therapies in musculoskeletal disorders are discussed at the cellular and molecule level.

https://www.mdpi.com/2227-9059/10/10/2477/pdf?version=1666602170

Role of Matrix Metalloproteinases in Musculoskeletal Diseases

Deficiency in the membrane cytoskeletal protein dystrophin is the underlying cause of the progressive muscle wasting disease named Duchenne muscular dystrophy. In order to detect novel disease marker candidates and confirm the complexity of the pathobiochemical signature of dystrophinopathy, mass spectrometric screening approaches represent ideal tools for comprehensive biomarker discovery studies. In this report, we describe the comparative proteomic analysis of young versus aged diaphragm muscles from wild type versus the dystrophic mdx-4cv mouse model of X-linked muscular dystrophy. The survey confirmed the drastic reduction of the dystrophin-glycoprotein complex in the mdx-4cv diaphragm muscle and concomitant age-dependent changes in key markers of muscular dystrophy, including proteins involved in cytoskeletal organization, metabolite transportation, the cellular stress response and excitation-contraction coupling. Importantly, proteomic markers of the regulation of membrane repair, tissue regeneration and reactive myofibrosis were detected by mass spectrometry and changes in key proteins were confirmed by immunoblotting. Potential disease marker candidates include various isoforms of annexin, the matricellular protein periostin and a large number of collagens. Alterations in these proteoforms can be useful to evaluate adaptive, compensatory and pathobiochemical changes in the intracellular cytoskeleton, myofiber membrane integrity and the extracellular matrix in dystrophin-deficient skeletal muscle tissues.

https://www.mdpi.com/2075-1729/12/11/1679/pdf?version=1666425413

Proteomic Identification of Markers of Membrane Repair, Regeneration and Fibrosis in the Aged and Dystrophic Diaphragm

Although progressive wasting and weakness of respiratory muscles are the prominent hallmarks of Duchenne muscular dystrophy (DMD) and long-COVID (also referred as the post-acute sequelae of COVID-19 syndrome); however, the underlying mechanism(s) leading to respiratory failure in both conditions remain unclear. We put together the latest relevant literature to further understand the plausible mechanism(s) behind diaphragm malfunctioning in COVID-19 and DMD conditions. Previously, we have shown the role of matrix metalloproteinase-9 (MMP9) in skeletal muscle fibrosis via a substantial increase in the levels of tumor necrosis factor-α (TNF-α) employing a DMD mouse model that was crossed-bred with MMP9-knockout (MMP9-KO or MMP9-/-) strain. Interestingly, recent observations from clinical studies show a robust increase in neopterin (NPT) levels during COVID-19 which is often observed in patients having DMD. What seems to be common in both (DMD and COVID-19) is the involvement of neopterin (NPT). We know that NPT is generated by activated white blood cells (WBCs) especially the M1 macrophages in response to inducible nitric oxide synthase (iNOS), tetrahydrobiopterin (BH4), and tetrahydrofolate (FH4) pathways, i.e., folate one-carbon metabolism (FOCM) in conjunction with epigenetics underpinning as an immune surveillance protection. Studies from our laboratory, and others researching DMD and the genetically engineered humanized (hACE2) mice that were administered with the spike protein (SP) of SARS-CoV-2 revealed an increase in the levels of NPT, TNF-α, HDAC, IL-1β, CD147, and MMP9 in the lung tissue of the animals that were subsequently accompanied by fibrosis of the diaphragm depicting a decreased oscillation phenotype. Therefore, it is of interest to understand how regulatory processes such as epigenetics involvement affect DNMT, HDAC, MTHFS, and iNOS that help generate NPT in the long-COVID patients.

/pdf/covid-19-mimics-pulmonary-dysfunction-in-muscular-dystrophy-1zbxx9f9.pdf

COVID-19 Mimics Pulmonary Dysfunction in Muscular Dystrophy as a Post-Acute Syndrome in Patients

Hyaluronic acid (HA) exerts a fundamental role in tissue repair. In vitro and animal studies demonstrated its ability to enhance wound healing. Nevertheless, in vivo human studies evaluating mechanisms involved in oral soft tissue repair are lacking. The aim of this study was to evaluate the in vivo effect of HA on early wound healing of human gingival (G) tissues.In the present randomized, split-mouth, double-blind, clinical trial, G biopsies were obtained in eight patients 24 h post-surgery after HA application (HA group) and compared with those obtained from the same patients without HA application (no treatment; NT group). Clinical response was evaluated through the Early Wound Healing Score (EHS). Microvascular density (MVD), collagen content and cellular proliferation were evaluated through sirius red and Masson trichrome staining, and Ki-67 immunohistochemistry, respectively. To assess collagen turnover, MMP-1, MMP-2, MMP-9, TGF-β1 protein levels and LOX, MMP1, TIMP1, TGFB1 gene expression were analyzed by western blot and real time polymerase chain reaction.Twenty-four hours after surgery, the EHS was significantly higher in the HA group. MVD, collagen content, and cell proliferation were not affected. LOX mRNA, MMP-1 protein, and TIMP1 gene expression were significantly upregulated in the HA compared to the NT group.The additional use of 0.8% HA gel does not modify new blood vessel growth in the early phase of gingival wound healing. Concerning the secondary outcomes, HA seems to enhance extracellular matrix remodeling and collagen maturation, which could drive early wound healing of G tissues to improve clinical parameters. 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/JPER.22-0338

Clinical, histological, immunohistochemical, and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: A randomized, split‐mouth trial

The three-dimensional multicellular spheroid (3D MCS) model has been employed in cholangiocarcinoma research as it generates 3D architecture and includes more physiological relevance with the multicellular arrangement. However, it is also essential to explain the molecular signature in this microenvironment and its structural complexity. The results indicated that poorly differentiated CCA cell lines were unable to form 3D MCS due to the lack of cell adhesion molecules with more mesenchymal marker expression. The well-differentiated CCA and cholangiocyte cell lines were able to develop 3D MCSs with round shapes, smooth perimeter, and cell adhesion molecules that led to the hypoxic and oxidative microenvironment detected. For MMNK-1, KKU-213C, and KKU-213A MCSs, the proteo-metabolomic analysis showed proteins and metabolic products altered compared to 2D cultures, including cell-cell adhesion molecules, energy metabolism-related enzymes and metabolites, and oxidative-related metabolites. Therefore, the 3D MCSs provide different physiological states with different phenotypic signatures compared to 2D cultures. Considering the 3D model mimics more physiological relevance, it might lead to an alternate biochemical pathway, targeting to improve drug sensitivity for CCA treatment. 

/pdf/the-impact-of-hypoxia-and-oxidative-stress-on-proteo-2p3yjnmg.pdf

The impact of hypoxia and oxidative stress on proteo-metabolomic alterations of 3D cholangiocarcinoma models

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.

/pdf/characterisation-of-progressive-skeletal-muscle-fibrosis-in-1c99dako.pdf

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior.

/pdf/mechanical-cues-e-cadherin-expression-and-cell-sociality-are-m7cfnxaq.pdf

Ambra Nava

Papers

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study

Mechanical Cues, E-Cadherin Expression and Cell “Sociality” Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior