Structural Musculotendinous Parameters That Predict Failed Tendon Healing After Rotator Cuff Repair.

Background: Healing of the rotator cuff after repair constitutes a major clinical challenge with reported high failure rates. Identifying structural musculotendinous predictors for failed rotator cuff repair could enable improved diagnosis and management of patients with rotator cuff disease. Purpose: To investigate structural predictors of the musculotendinous unit for failed tendon healing after rotator cuff repair. Study Design: Cohort study; Level of evidence, 2. Methods: Included were 116 shoulders of 115 consecutive patients with supraspinatus (SSP) tear documented on magnetic resonance imaging (MRI) who were treated with an arthroscopic rotator cuff repair. Preoperative assessment included standardized clinical and imaging (MRI) examinations. Intraoperatively, biopsies of the joint capsule, the SSP tendon, and muscle were harvested for histological assessment. At 3 and 12 months postoperatively, patients were re-examined clinically and with MRI. Structural and clinical predictors of healing were evaluated using logistic and linear regression models. Results: Structural failure of tendon repair, which was significantly associated with poorer clinical outcome, was associated with older age (ß = 1.12; 95% CI, 1.03 to 1.26; P = .03), shorter SSP tendon length (ß = 0.89; 95% CI, 0.8 to 0.98; P = .02), and increased proportion of slow myosin heavy chain (MHC)-I/fast MHC-II hybrid muscle fibers (ß = 1.23; 95% CI, 1.07 to 1.42; P = .004). Primary clinical outcome (12-month postoperative Constant score) was significantly less favorable for shoulders with fatty infiltration of the infraspinatus muscle (ß = -4.71; 95% CI, -9.30 to -0.12; P = .044). Conversely, a high content of fast MHC-II muscle fibers (ß = 0.24; 95% CI, 0.026 to 0.44; P = .028) was associated with better clinical outcome. Conclusion: Both decreased tendon length and increased hybrid muscle fiber type were independent predictors for retear. Clinical outcome was compromised by tendon retearing and increased fatty infiltration of the infraspinatus muscle. A high content of fast MHC-II SSP muscle fibers was associated with a better clinical outcome. Registration: NCT02123784 (ClinicalTrials.govidentifier).

Exploring the Role of Osteosarcoma-Derived Extracellular Vesicles in Pre-Metastatic Niche Formation and Metastasis in the 143-B Xenograft Mouse Osteosarcoma Model.

The pre-metastatic niche (PMN) is a tumor-driven microenvironment in distant organs that can foster and support the survival and growth of disseminated tumor cells. This facilitates the establishment of secondary lesions that eventually form overt metastasis, the main cause of cancer-related death. In recent years, tumor-derived extracellular-vesicles (EVs) have emerged as potentially key drivers of the PMN. The role of the PMN in osteosarcoma metastasis is poorly understood and the potential contribution of osteosarcoma cell-derived EVs to PMN formation has not been investigated so far. Here, we characterize pulmonary PMN development using the spontaneously metastasizing 143-B xenograft osteosarcoma mouse model. We demonstrate the accumulation of CD11b+ myeloid cells in the pre-metastatic lungs of tumor-bearing mice. We also establish that highly metastatic 143-B and poorly metastatic SAOS-2 osteosarcoma cell-derived EV education in naïve mice can recapitulate the recruitment of myeloid cells to the lungs. Surprisingly, despite EV-induced myeloid cell infiltration in the pre-metastatic lungs, 143-B and SAOS-2 EVs do not contribute towards the 143-B metastatic burden in the context of both spontaneous as well as experimental metastasis in severe-combined immunodeficient (SCID) mice. Taken together, OS-derived EVs alone may not be able to form a functional PMN, and may perhaps require a combination of tumor-secreted factors along with EVs to do so. Additionally, our study gives a valuable insight into the PMN complexity by providing the transcriptomic signature of the premetastatic lungs in an osteosarcoma xenograft model for the first time. In conclusion, identification of regulators of cellular and molecular changes in the pre-metastatic lungs might lead to the development of a combination therapies in the future that interrupt PMN formation and combat osteosarcoma metastasis.

Comparative Degradomics of Porcine and Human Wound Exudates Unravels Biomarker Candidates for Assessment of Wound Healing Progression in Trauma Patients.

Impaired cutaneous wound healing is a major complication in elderly people and patients suffering from diabetes, the rate of which is rising in industrialized countries. Heterogeneity of clinical manifestations hampers effective molecular diagnostics and decisions for appropriate therapeutic regimens. Using a customized positional quantitative proteomics workflow, we have established a time-resolved proteome and N-terminome resource from wound exudates in a clinically relevant pig wound model that we exploited as a robust template to interpret a heterogeneous dataset from patients undergoing the same wound treatment. With zyxin, IQGA1, and HtrA1, this analysis and validation by targeted proteomics identified differential abundances and proteolytic processing of proteins of epidermal and dermal origin as prospective biomarker candidates for assessment of critical turning points in wound progression. Thus, we show the possibility of using a fine-tuned animal wound model to bridge the translational gap as a prerequisite for future extended clinical studies with large cohorts of individuals affected by healing impairments. Data are available via ProteomeXchange with identifier PXD006674.

Time-Resolved Analysis of Matrix Metalloproteinase Substrates in Complex Samples.

Identification of physiological substrates is the key to understanding the pleiotropic functions of matrix metalloproteinases (MMPs) in health and disease. Quantitative mass spectrometry-based proteomics has revolutionized current approaches in protease substrate discovery and helped to unravel many new MMP activities in complex biological systems. Multiplexing further extended the capabilities of these techniques and facilitated more complicated experimental designs that include multiple proteases or monitoring the activity of a single protease at more than one concentration or at multiple time points with a complex test proteome. In this chapter, we provide a protocol for time-resolved iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS), with the focus on MMP substrate identification and characterization in cell culture supernatants and introduce an automated procedure for the interpretation of time-resolved iTRAQ-TAILS datasets.

In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.

Time-resolved analysis of the matrix metalloproteinase 10 substrate degradome.

Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.