Extracellular Matrix Damage Research Articles

Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin

Ethnopharmacological relevancePerilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. Aim of the studyThe present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Materials and methodsHuman dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. ResultsPLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. ConclusionOur results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging.

Extracellular Matrix Proteolysis by MT1-MMP Contributes to Influenza-Related Tissue Damage and Mortality.

Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival.We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.

Disruption of mechanical stress in extracellular matrix is related to Stanford type A aortic dissection through down-regulation of Yes-associated protein.

In this study, we assessed whether the down-regulation of Yes-associated protein (YAP) is involved in the pathogenesis of extracellular matrix (ECM) mechanical stress-induced Stanford type A aortic dissection (STAAD). Human aortic samples were obtained from heart transplantation donors as normal controls and from STAAD patients undergoing surgical replacement of the ascending aorta. Decreased maximum aortic wall velocity, ECM disorders, increased VSMC apoptosis, and YAP down-regulation were identified in STAAD samples. In a mouse model of STAAD, YAP was down-regulated over time during the development of ECM damage, and increased VSMC apoptosis was also observed. YAP knockdown induced VSMC apoptosis under static conditions in vitro, and the change in mechanical stress induced YAP down-regulation and VSMC apoptosis. This study provides evidence that YAP down-regulation caused by the disruption of mechanical stress is associated with the development of STAAD via the induction of apoptosis in aortic VSMCs. As STAAD is among the most elusive and life-threatening vascular diseases, better understanding of the molecular pathogenesis of STAAD is critical to improve clinical outcome.

EPV 6. Value of transcranial brain stem sonography in the diagnosis and prognosis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease, most common in adult-onset, characterized by a progressive failure of upper and lower motor neurons. Symptoms of lower motor neuron disease such as progressive atrophic weakness may obscure upper motor neuron signs so that the correct diagnosis and initiation of treatment may be delayed. The aim of the study was to evaluate the usefulness of transcranial brain stem sonography (TCS) as additional diagnostic tool in ALS and discussed the prognostic value. 94 patients with sporadic ALS and 46 age- and gender- matched healthy controls were studied with TCS according to a standardized protocol used in Parkinson patients. The observed hyperechogenic changes in the mesencephalon were categorized depending on their size into three groups („clear“ > 0.25 cm[b], „possible“ < 0.25 cm[b], „no”). 48% of the ALS-patients and 2% of healthy controls showed a clear mesencephalic hyperechogenic structure (p < 0,001). 20% of ALS-patients and 4% controls showed possible and 24% ALS-patients and 78% showed no hyperechogenic structures in the brainstem. TCS findings did not correlate with gender, disease onset (spinal, bulbar), disease duration, disease severity (ALSFRS-R), motor evoked potentials (MEP), and MRT results. TCS could serve as an additional diagnostic tool in ALS in order to improve early diagnosing. We detected significantly more hyperechogenicities in the anatomical area of the pyramidal fibre tract in the mesencephalon of ALS patients compared to healthy controls. Although the neuropathological correlate is unclear, the observed changes reflect upper motor neuron degeneration in pyramidal tract, proliferation of glial cells and extracellular matrix damage. To assess the value of TCS as a diagnostic and prognostic marker in ALS, further cross-sectional and longitudinal studies are necessary, especially retrospective observation of disease-progression. This study was supported by a grant to JG by the BMBF through the E-RARE framework and in collaboration with the German Network for Motoneuron Diseases (MND-NET).

Peroxynitrite-mediated oxidation of plasma fibronectin

Fibronectin is a large dimeric glycoprotein present in both human plasma and in basement membranes. The latter are specialized extracellular matrices underlying endothelial cells in the artery wall. Peroxynitrous acid (ONOOH) a potent oxidizing and nitrating agent, is formed in vivo from superoxide and nitric oxide radicals by stimulated macrophages and other cells. Considerable evidence supports ONOOH involvement in human atherosclerotic lesion development and rupture, possibly via extracellular matrix damage. Here we demonstrate that Tyr and Trp residues on human plasma fibronectin are highly sensitive to ONOOH with this resulting in the formation of 3-nitrotyrosine, 6-nitrotryptophan and dityrosine as well as protein aggregation and fragmentation. This occurs with equimolar or greater levels of oxidant, and in a dose-dependent manner. Modification of Tyr was quantitatively more significant than Trp (9.1% versus 1.5% conversion with 500μM ONOOH) after accounting for parent amino acid abundance, but only accounts for a small percentage of the total oxidant added. LC-MS studies identified 28 nitration sites (24 Tyr, 4 Trp) with many of these present within domains critical to protein function, including the cell-binding and anastellin domains. Human coronary artery endothelial cells showed decreased adherence and cell-spreading on ONOOH-modified fibronectin compared to control, consistent with cellular dysfunction induced by the modified matrix. Studies on human atherosclerotic lesions have provided evidence for co-localization of 3-nitrotyrosine and fibronectin. ONOOH-mediated fibronectin modification and compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the fibrous cap of atherosclerotic lesions, and an increased propensity to rupture.

Melatonin protects podocytes from angiotensin II-induced injury in an invitro diabetic nephropathy model.

Diabetic nephropathy (DN) is a severe renal disorder characterized by podocyte damage and accumulation of extracellular matrix leading to further glomerulosclerosis, possibly via the activation of inflammatory signaling and the generation of oxidative stress. Melatonin has been considered a robust anti‑oxidant, and is able to attenuate DN in several animal models. In the present study, cell viability was examined by CCK-8 assay. Cell apoptosis, intracellular reactive oxygen species and mitochondrial membrane potential (MMP) levels were measured by flow cytometry. Western blot analysis was performed to detect the expression of Nephrin, Bcell lymphoma‑2 (Bcl‑2)‑associatedXprotein (Bax), Bcl-2, Caspase-3, Janus kinase (JAK)2 and Signal transducer and activator of transcription (STAT)3 in podocytes. Based on an invitro podocyte injury model, the present study found that the application of melatonin significantly reduced AngII‑induced apoptosis and increased the proliferative rate of cells, as evidenced by decreased expression levels of apoptotic proteins, including Caspase‑3 and Bax, and a change in the ratio of Bax/Bcl‑2. Further investigation demonstrated that a reduction in oxidative stress and the recovery of mitochondrial function were involved in this protective effect. In addition, the Jak/STAT signaling pathway was inhibited, indicating that cytokine‑mediated inflammation was also targeted by melatonin. The present study demonstrated for the first time, to the best our knowledge, that melatonin exerted an anti-apoptotic effect in AngII-mediated podocyte injury, and indicates a possible mechanism to explain the protective effect of melatonin in DN.

Chemoprotective effect of omega-3 fatty acids on thioacetamide induced hepatic fibrosis in male rats.

The current study was designed to investigate the possible protective effect of omega-3 fatty acids from fish oil on hepatic fibrosis induced by thioacetamide (TAA) in male rats. The experimental animals were divided into four groups. The first group was received saline solution and served as control. The second group was given 250 mg/kg body weight of TAA. The third group was treated with omega-3 fatty acids and TAA. The fourth group was given saline solution and supplemented with omega-3 fatty acids. Treatment of rats with TAA for three and six weeks resulted in a significant decrease in body weight gain, while the value of liver/body weight ratio was statistically increased. Furthermore, the levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase and total bilirubin were significantly increased. After three weeks of exposure to only TAA, liver sections showed an abnormal morphology characterized by noticeable fibrosis with the extracellular matrix collagen contents and damage of liver cells’ structure. Liver sections from rats treated with only TAA for six weeks revealed an obvious increase in extracellular matrix collagen content and bridging fibrosis. Treating TAA-intoxicated rats with omega-3 fatty acids significantly attenuated the severe physiological and histopathological changes. Finally, the present investigation suggests that omega-3 fatty acids could act against hepatic fibrosis induced by TAA due to its antioxidant properties, thus supporting its use in hepatic fibrosis therapy.

Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin

Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to BM was characterized for isolated murine BM, and purified murine laminin-111. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Endothelial cell adhesion to isolated laminin-111 exposed to 10μM or higher levels of ONOOH was significantly decreased (~25%) compared to untreated controls. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture.

OP28 - Laminin, a key component of tissue extracellular matrix, is a major target for peroxynitrous acid

Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to laminin was characterized with purified murine laminin-111, and murine BM extracts containing multiple matrix components. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. This damage was modulated by bicarbonate, a known modifier of ONOOH reactions. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Significant damage was detected with equimolar or greater concentrations of ONOOH. Endothelial cell adhesion to isolated laminin-111 was significantly decreased (~25%) compared to controls, on exposure to 10 μM or higher levels of ONOOH. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture.

The Temporal Pattern of Changes in Serum Biomarker Levels Reveals Complex and Dynamically Changing Pathologies after Exposure to a Single Low-Intensity Blast in Mice.

Time-dependent changes in blood-based protein biomarkers can help identify the ­pathological processes in blast-induced traumatic brain injury (bTBI), assess injury severity, and monitor disease progression. We obtained blood from control and injured mice (exposed to a single, low-intensity blast) at 2-h, 1-day, 1–week, and 1-month post-injury. We then determined the serum levels of biomarkers related to metabolism (4-HNE, HIF-1α, ceruloplasmin), vascular function (AQP1, AQP4, VEGF, vWF, Flk-1), inflammation (OPN, CINC1, fibrinogen, MIP-1a, OX-44, p38, MMP-8, MCP-1 CCR5, CRP, galectin-1), cell adhesion and the extracellular matrix (integrin α6, TIMP1, TIMP4, Ncad, connexin-43), and axonal (NF-H, Tau), neuronal (NSE, CK-BB) and glial damage (GFAP, S100β, MBP) at various post-injury time points. Our findings indicate that the exposure to a single, low-intensity blast results in metabolic and vascular changes, altered cell adhesion, and axonal and neuronal injury in the mouse model of bTBI. Interestingly, serum levels of several inflammatory and astroglial markers were either unchanged or elevated only during the acute and subacute phases of injury. Conversely, serum levels of the majority of biomarkers related to metabolic and vascular functions, cell adhesion, as well as neuronal and axonal damage remained elevated at the termination of the experiment (1 month), indicating long-term systemic and cerebral alterations due to blast. Our findings show that the exposure to a single, low-intensity blast induces complex pathological processes with distinct temporal profiles. Hence, monitoring serum biomarker levels at various post-injury time points may provide enhanced diagnostics in blast-related neurological and multi-system deficits.

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.

Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise.

ABSTRACTThe role of inflammation in tendon injury is uncertain and a topic of current interest. In vitro studies of tendon accelerated overload damage can serve as a valuable source of information on the early stages of tendinopathy. Viable fascicle bundles from bovine flexor tendons were subjected to cyclic uniaxial loading from 1–10% strain. Immuno‐staining for inflammatory markers and matrix degradation markers was performed on the samples after mechanical testing. Loaded samples exhibited visible extracellular matrix damage, with disrupted collagen fibers and fiber kinks, and notable damage to the interfascicular matrix. Inflammatory markers COX‐2 and IL‐6 were only expressed in the cyclically loaded samples. Collagen degradation markers MMP‐1 and C1,2C were colocalized in many areas, with staining occurring in the interfascicular matrix or the fascicular tenocytes. These markers were present in control samples, but staining became increasingly intense with loading. Little MMP‐3 or MMP‐13 was evident in control sections. In loaded samples, some sections showed intense staining of these markers, again localized to interfascicular regions. This study suggests that inflammatory markers may be expressed rapidly after tendon overload exercise. Interestingly, both inflammation and damage‐induced matrix remodeling seem to be concentrated in, or in the vicinity of, the highly cellular interfascicular matrix. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 33:889–897, 2015.

Xenotransplantation literature update, January-February 2015.

Xenotransplantation literature update, January-February 2015.

Hypohalous acids contribute to renal extracellular matrix damage in experimental diabetes.

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid–derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1β1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W28 and W192 are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid–derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.

Functional Recovery of Axonal Injury Induced by Gelatin-Hydrogel Film and PRP: An Initial Study in Rats

In today’s society, axonal regeneration is considered very important because of high incidence of nerve injuries. The injury may cause vast damage of extracellular matrix leading to the condition wherein cell lost their environment to live. In this situation, new strategies by the introduction of scaffold to function as synthetic extracellular matrix are needed. In the field of tissue engineering, a large variety of carriers and matrices have been utilized as a scaffold to direct axonal growth. In this study, a new strategy by the combined application of gelatin hydrogel film and Platelet Rich Plasma (PRP) to induce functional recovery of sciatic nerve crushed injury was investigated in Wistar rat model. Assessment of motorist and sensory functions were done to evaluate the recovery processes. It was found that the application of gelatin hydrogel film with controlled release manner combined with PRP results faster axonal regeneration. Further research by different injury model and other investigation factors are needed to confirm the effectiveness of the strategy.

Nrf2 null enhances UVB-induced skin inflammation and extracellular matrix damages.

Nrf2 plays a critical role in defending against oxidative stress and inflammation. We previously reported that Nrf2 confers protection against ultraviolet-B (UVB)-induced inflammation, sunburn reaction, and is involved in sulforaphane-mediated photo-protective effects in the skin. In this study, we aimed to demonstrate the protective role of Nrf2 against inflammation-mediated extracellular matrix (ECM) damage induced by UVB irradiation. Ear biopsy weights were significantly increased in both Nrf2 wild-type (Nrf2 WT) and knockout (Nrf2 KO) mice one week after UVB irradiation. However, these weights increased more significantly in KO mice compared to WT mice, suggesting a greater inflammatory response in KO mice. In addition, we analyzed the protein expression of numerous markers, including macrophage inflammatory protein-2 (MIP-2), pro-matrix metalloproteinase-9 (MMP-9), and p53. p53, a regulator of DNA repair, was overexpressed in Nrf2 KO mice, indicating that the absence of Nrf2 led to more sustained DNA damage. There was also more substantial ECM degradation and increased inflammation in UVB-irradiated Nrf2 KO mice compared to UVB-irradiated WT mice. Furthermore, the protective effects of Nrf2 in response to UVB irradiation were mediated by increased HO-1 protein expression. Collectively, our results show that Nrf2 plays a key role in protecting against UVB irradiation and that the photo-protective effect of Nrf2 is closely related to the inhibition of ECM degradation and inflammation.

EP-1814: Study of radiation-induced damage and remodeling of extracellular matrix of rectum and bladder by optical methods

EP-1814: Study of radiation-induced damage and remodeling of extracellular matrix of rectum and bladder by optical methods

Rheological and Dynamic Integrity of Simulated Degenerated Disc and Consequences After Cross-linker Augmentation

An in situ study using whole-organ culture system. To study the effect of disc degeneration at different stages on its rheological and dynamic properties and to investigate the efficacy of exogenous cross-linking therapy. Disc degeneration can involve protein denaturation or microdefects to the disc's collagen fiber network. A disc degeneration model using whole-organ culture technique can be effectively used for the screening of treatments of degenerated discs. Exogenous cross-linking therapy has been shown to enhance the mechanical properties of the disc by cross-linking collagen. However, the efficacy of this therapy on the degenerated disc is unclear. A total of 40 porcine thoracic discs were assigned to 5 groups: intact discs, moderately degenerated discs, moderately degenerated discs with cross-linker augmentation, severely degenerated discs, and severely degenerated discs with cross-linker augmentation. The disc degeneration was simulated by trypsin digestion and mechanical fatigue loading. Rheological properties, dynamic properties, water content, and histological analysis were conducted after a 7-day incubation. The mechanical properties of moderate degenerated discs significantly decrease both in rheological and dynamic properties, and laminate structure disorganization was observed. Mechanical defects of severely degenerated discs resulted in disc height loss, an increase in the aggregate modulus and stiffness modulus, and a decrease in the damping coefficient, hydraulic permeability, and water content. Cross-linker augmentation significantly recovered mechanical properties of moderately degenerated discs and restored the water content compared with the intact disc. However, the augmentation did not fully repair the severely degenerated discs. Trypsin-induced extracellular matrix damage resulted in a change of the disc's biomechanics. Cross-linker augmentation recovers the rheological and dynamic properties of moderately degenerated discs but not of the severely degenerated discs. The genipin cross-linker may be able to improve the proteoglycan depletion effect in the nucleus pulposus but may not be effective to restore the structural damage in the collagen molecule of the anulus fibrosus.

Tendon extracellular matrix damage detection and quantification using automated edge detection analysis

The accumulation of sub-rupture tendon fatigue damage in the extracellular matrix, particularly of type I collagen fibrils, is thought to contribute to the development of tendinopathy, a chronic and degenerative pathology of tendons. Quantitative assessment of collagen fibril alignment is paramount to understanding the importance of matrix injury to cellular function and remodeling capabilities. This study presents a novel application of edge detection analysis to calculate local collagen fibril orientation in tendon. This technique incorporates damage segmentation and stratification by severity which will allow future analysis of the direct effect of matrix damage severity on the cellular and molecular response.

Novel Proteolytic Microvesicles Released from Human Macrophages after Exposure to Tobacco Smoke

Cigarette smoking damages the extracellular matrix in a variety of locations, leading to atherosclerotic plaque instability and emphysematous lung destruction, but the underlying mechanisms remain poorly understood. Here, we sought to determine whether exposure of human macrophages, a key participant in extracellular matrix damage, to tobacco smoke extract (TSE) induces the release of microvesicles (MVs; or microparticles) with proteolytic activity; the major proteases involved; and the cellular mechanisms that might mediate their generation. We found that MVs released from TSE-exposed macrophages carry substantial gelatinolytic and collagenolytic activities that surprisingly can be predominantly attributed to a single transmembrane protease of the matrix metalloproteinase (MMP) superfamily (namely, MMP14). Flow cytometric counts revealed that exposure of human macrophages to TSE for 20 hours more than quadrupled their production of MMP14-positive MVs (control, 1112 ± 231; TSE-induced, 5823 ± 2192 MMP14-positive MVs/μL of conditioned medium; means ± SEM; n = 6; P < 0.01). Our results indicate that the production of these MVs by human macrophages relies on a series of regulated steps that include activation of two mitogen-activated protein kinases (MAPKs, i.e., the Jun N-terminal kinase and p38 MAPK), and then MAPK-dependent induction and maturation of cellular MMP14, a remarkable accumulation of MMP14 into nascent plasma membrane blebs, and finally caspase- and MAPK-dependent apoptosis and apoptotic microvesicle generation. Proteolytically active MVs induced by tobacco smoke may be novel mediators of clinical important matrix destruction in smokers.