Collagen Cross-Linking in Tendons: The Role of Lysyl Oxidase and Posttranslational Modifications in Injury, Aging, and Congenital Disorders.

The purpose of this study was to investigate the in vivo material and mechanical properties of the human Achilles tendon in the presence of tendinopathy. Real-time ultrasound imaging and dynamometry were used to assess Achilles tendon stiffness, Young's modulus, stress, strain, and cross-sectional area (CSA) in 12 individuals with Achilles tendinopathy and 12 age- and gender-matched controls. The results of this study suggest that tendinopathy weakens the mechanical and material properties of the tendon. Tendinopathic tendons had greater CSA, lower tendon stiffness, and lower Young's modulus. These alterations in mechanical characteristics may put the Achilles tendon at a higher risk to sustain further injury and prolong the time to recovery. Results from this study may be used to design treatment strategies that specifically target these deficits, leading to faster and permanent recovery from tendinopathy.

Because of its dynamic nature, the composition and structure of the myocardial collagen network can be reversibly modified to adapt to transient cardiac injuries. In response to persistent injury, however, irreversible, maladaptive changes of the network occur leading to fibrosis, mostly characterized by the excessive interstitial and perivascular deposition of collagen types I and III fibers. It is now becoming apparent that myocardial fibrosis directly contributes to adverse myocardial remodeling and the resulting alterations of left ventricular (LV) anatomy and function present in the major types of cardiac diseases. The enzyme lysyl oxidase (LOX) is a copper-dependent extracellular enzyme that catalyzes lysine-derived cross-links in collagen and elastin. LOX-mediated cross-linking of collagen types I and III fibrils leads to the formation of stiff collagen types I and III fibers and their subsequent tissue deposition. Evidence from experimental and clinical studies shows that the excess of LOX is associated with an increased collagen cross-linking and stiffness. It is thus conceivable that LOX upregulation and/or overactivity could underlie myocardial fibrosis and altered LV mechanics and contribute to the compromise of LV function in cardiac diseases. This review will consider the molecular aspects related to the regulation and actions of LOX, namely, in the context of collagen synthesis. In addition, it will address the information related to the role of myocardial LOX in heart failure and the potential benefits of controlling its expression and function.

Gastrocnemius medialis muscle-tendon properties and their relationship with power and reactive strength in elite youth soccer players

Subject-specific finite element analysis to characterize the influence of geometry and material properties in Achilles tendon rupture

Novel noninvasive imaging modalities such as quantitative magnetic resonance imaging (qMRI) and shear wave elastography (SWE) allow for assessment of soft tissue microstructure and composition, which ultimately may be associated with functional and material properties. To provide a narrative review of the scientific techniques and clinical applications of qMRI and SWE for the evaluation of soft tissue about the knee and shoulder, including the meniscus, the anterior cruciate ligament (ACL), and the rotator cuff. Review. A literature search was performed in October 2022 via PubMed using the following keywords: "quantitative MRI tendon," quantitative MRI ligament,""quantitative MRI cartilage," or "shear wave elastography tendon." Only articles related to clinical applications were included in this review. Conventional imaging techniques, including standard morphologic magnetic resonance imaging (MRI) and ultrasound imaging, have limited ability to evaluate the material and functional properties of soft tissue; qMRI builds on the limitations of conventional morphologic MRI by allowing for detection of early articular cartilage changes, differentiation of healed versus unhealed meniscal tissue, and quantification of ACL graft maturity. SWE can evaluate the material properties of rotator cuff and Achilles tendons after injury, which may provide insight into both the chronicity and the healing status of the aforementioned injuries. Our review of the literature showed that quantitative imaging techniques, including qMRI and SWE, may both improve early detection of pathology and aid in comprehensive evaluation after treatment.

Microassay for the assessment of low levels of hydroxyproline.

Background and Aims We studied the downstream and mechanism of β-arrestins signaling in renal fibrosis process and the role of lysyl oxidase (LOX) in the AT1R-β-arrestins pathway. Method The mechanism of β-arrestins signaling was studied in normal rat kidney tubule epithelial cells (NRK-52E) treated with SII in vitro. BAPN or placebo was administered during ischemia reperfusion (IR)-induced fibrosis progression. Collagen crosslinking and fibrosis progression were assessed histologically and biochemically. Results The mRNA and protein levels of β-arrestin-1 and β-arrestin-2 were significantly upregulated in renal fibrosis model both in vitro and in vivo. SII activated the ERK-STAT3 PY705 but not STAT3-Try727 in nucleus of NRK-52E cells, which effects were abolished when transfection of siRNA targeting β-arrestin-1 and β-arrestin-2 or pretreated with PD98059 (MEK inhibitor). LOX was strongly induced in fibrotic kidney and NRK-52E cells treated with SII. Active LOX significantly increased collagen crosslinking. In established IR-28d renal fibrosis, LOX inhibition promoted fibrosis reversal and with a 25% decrease insoluble collagen. Gene silencing of β-arrestin-1 + 2 or STAT3 apparently inhibited SII-induced LOX expression in vitro. Besides, chromatin immunoprecipitation (ChIP) assay clearly demonstrating the interaction between STAT3 and the LOX promoter, which indicated LOX is a direct target gene of SII-β-arrestins-STAT3 signaling. Conclusion The ERK/STAT3 was downstream of AT1R-β-arrestins, ERK entered the nucleus and activated STAT3-PY705. LOX mediates collagen crosslinking and fibrotic matrix stabilization during renal fibrosis via the AT1R-β-arrestins-ERK-STAT3-PY705 signaling. By blocking this profibrotic pathway, therapeutic LOX inhibition attenuates the fibrosis and suggesting target the LOX has significant potency for the treatment of patients with fibrotic kidney disorders.

Purpose: Lysyl oxidase (LOX) increases extracellular matrix stiffness by cross-linking collagen. A stiffer matrix drives integrin activation, and therefore LOX is believed to promote cancer cell invasion through enhanced integrin signaling. However increased matrix stiffness does not necessarily lead to a more permissive environment for invasion. Fibrillar collagen matrix orientation, density and porosity also determine how well cells can invade. Assays based on collagen gel-covered transwell systems have previously been used to show that LOX is required for invasion. These systems are driven by a chemoattractant gradient, and thus a role for LOX in chemotaxis could not be excluded. In this study we aimed to clarify whether LOX was required for matrix invasion or chemotaxis. Experimental procedures: To set up a 3D invasion assay that was not driven by a chemoattractant gradient, spheroids from multiple LOX expressing cancer cell lines were embedded into a thick 3D collagen gel. Cells were then allowed to randomly invade into the surrounding collagen. Alternatively, cancer cells were allowed to invade a fibroblast-modified 3D collagen gel in an organotypic assay. In this assay the invasion was driven by a strong chemoattractant gradient through a dense collagen gel, with cancer cells cultured atop the collagen gel at the air-medium interface. A Dunn chemotaxis chamber or a transwell tissue culture insert was used to investigate chemotaxis towards serum or EGF. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) specific to human LOX was used to deplete LOX expression in the cancer cells, to test potential LOX functions in the above assays. Results: LOX depletion in parental MDA-MB-231 cells, MDA-MB-231 brain or bone metastatic sub-population cells, U87 and U118 cells did not impact 3D collagen gel invasion. LOX inhibition by BAPN also did not impact collagen gel invasion in MDA-MB-231, LN229, U87, U118 and U138 cells. The data indicated that LOX was not required for invasion through a thick 3D collagen gel in vitro. However, in the chemotactic-driven organotypic assay, loss of LOX in MDA-MB-231 cells completely abolished invasion. When the chemotactic potential towards serum and EGF of LOX depleted MDA-MB-231 and U87 cells was tested using Dunn chemotaxis chambers, we observed strong chemotactic defects in both cell lines. The chemotactic defects of these cells were also observed when a transwell based chemotaxis assay was used. Random cell migration of MDA-MB-231 and U87 cells were otherwise not affected by LOX depletion. Conclusions: LOX is not required for collagen matrix invasion per se. Instead LOX is important for cells to sense chemoattractants and to maintain directional cell migration. The molecular mechanisms underlying these observations are now the subject of further investigation. Citation Format: Haoran Tang, Nathalie Dhomen, Richard Marais. Lysyl oxidase is required for chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2017-905

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by low bone mass and spontaneous fractures, as well as extra-skeletal manifestations, such as dental abnormalities, blue sclera, hearing loss and joint hypermobility. Tendon ruptures have been reported in OI patients. Here, we characterized the biomechanical, structural and tissue material properties of bone and tendon in 5-week-old female osteogenesis imperfecta mice (oim), a validated model of severe type III OI, and compared these data with age- and sex-matched WT littermates. Oim tendons were less rigid and less resistant than those of WT mice. They also presented a significantly higher rate of pentosidine, without significant modification of enzymatic crosslinking. The oim bones were less resistant and avulsion fractures were evident at high tendinous stress areas. Alterations of trabecular and cortical bone microarchitectures were noticed in young female oim. Bone tissue material properties were also modified, with a less mature and more mineralized matrix in association with lower collagen maturity. Our data suggest that the tendon-to-bone unit is affected in young oim mice, which could explain tendon ruptures and bone fragility observed in OI patients.

LOX From Salivary Adenoid Cystic Carcinoma-Associated Fibroblast Promotes Fibrosis in the Pulmonary Pre-metastatic Niche

Purpose: Lysyl oxidase (LOX) stabilizes the extracellular matrix (ECM) by cross-linking collagen and elastin molecules. In proliferative diabetic retinopathy (PDR), there is ECM remodeling with neovascularization and basement membrane changes. While protease activities are well reported, the role of LOX in the pathogenesis of diabetic retinopathy is less studied. This study was done to see the effect of high glucose on the activity and expression of LOX and its isoforms in ARPE-19 cells.Materials and methods: ARPE-19 cells were exposed to high glucose up to 48 h, and LOX activity was determined by N-acetyl-3,7-dihydroxyphenoxazine assay. The mRNA expression of LOX and its isoforms was done by real-time PCR and the protein expression by ELISA. Immunohistochemistry for LOX was done in epiretinal membrane from PDR.Results: With an increase in glucose concentration LOX activity and protein was reduced significantly at 30 mM glucose at 48 h. mRNA expression of LOX, LOXL1, and LOXL2 varied with time and concentration of glucose. Vascular endothelial growth factor (VEGF) increased the LOX activity as well as the mRNA expression. Pigment epithelium-derived factor (PEDF) downregulated the mRNA expression of LOX, LOXL1, and LOXL2. The matrix metalloprotease (MMP) activity increased significantly with the increase in glucose concentration. The diabetic neovascular membrane showed increased immunostaining of LOX.Conclusions: This study suggests that although the LOX activity, which is composite of all the isoforms, was reduced under high glucose conditions, there was a differential mRNA expression with increased LOX and LOXL1 and decreased LOXL2 expression.

Lysyl oxidase (LOX) controls matrix remodeling, a key process that underlies cardiovascular diseases and heart failure; however, a lack of suitable animal models has limited our knowledge with regard to the contribution of LOX to cardiac dysfunction. Here, we assessed the impact of LOX overexpression on ventricular function and cardiac hypertrophy in a transgenic LOX (TgLOX) mouse model with a strong cardiac expression of human LOX. TgLOX mice exhibited high expression of the transgene in cardiomyocytes and cardiofibroblasts, which are associated with enhanced LOX activity and H2O2 production and with cardiofibroblast reprogramming. LOX overexpression promoted an age-associated concentric remodeling of the left ventricle and impaired diastolic function. Furthermore, LOX transgenesis aggravated angiotensin II (Ang II)-induced cardiac hypertrophy and dysfunction, which triggered a greater fibrotic response that was characterized by stronger collagen deposition and cross-linking and high expression of fibrotic markers. In addition, LOX transgenesis increased the Ang II-induced myocardial inflammatory infiltrate, exacerbated expression of proinflammatory markers, and decreased that of cardioprotective factors. Mechanistically, LOX overexpression enhanced oxidative stress and potentiated the Ang II-mediated cardiac activation of p38 MAPK while reducing AMPK activation. Our findings suggest that LOX induces an age-dependent disturbance of diastolic function and aggravates Ang II-induced hypertrophy, which provides novel insights into the role of LOX in cardiac performance.-Galán, M., Varona, S., Guadall, A., Orriols, M., Navas, M., Aguiló, S., de Diego, A., Navarro, M. A., García-Dorado, D., Rodríguez-Sinovas, A., Martínez-González, J., Rodriguez, C. Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II-induced hypertrophy.

Lysyl oxidase (LOX) is necessary for the biosynthesis of functional collagen through its crosslinking of collagen and elastin. Increased LOX expression has been associated with hepatic, lung and kidney fibrosis, but the role of LOX in the heart is not known. Cardiac fibroblasts constitutively express pro‐LOX protein and bone morphogenic protein‐1, which cleaves pro‐LOX to generate active LOX and LOX‐propeptide. We hypothesized that LOX expression and activity in the heart is regulated by estrogen and may contribute to gender specific cardioprotection by altering collagen processing. Adult cardiac fibroblasts were isolated from ovariectomized (OX) and intact female rats. Cells were grown in media with 10% FBS, and serum starved prior to treatment with 17β‐estradiol. Fibroblasts from OX rats had the highest basal expression of pro‐LOX (200% vs. intact), which was suppressed by estrogen. In contrast, estrogen enhanced pro‐ and active LOX expression in fibroblasts from intact females. These findings indicate that changes in hormonal status alter cardiac fibroblast expression of pro‐form and active LOX. Future studies are warranted to evaluate the estrogenic modulation of LOX expression and activity in the heart, as well as the resulting effects on myocardial collagen and cardiac function.

Object: Lysyl oxidase (LOX) family is the extracellular matrix-modifying enzymes comprising of LOX and LOX-like (LOXL) 1-4. These enzymes have intracellular functions involved in the regulation of motility, migration, differentiation, and transcription in various types of cells. LOX is associated with mesenchymal reconstruction that is essential for cancer invasion and metastasis. LOX overexpression is crucial to promoting tumor growth and metastasis in several cancers such as breast cancer and colorectal cancer, however, role of LOX in gastric cancer remain unknown. Here, we investigated the clinicopathological significance of LOX and LOXL2 in gastric cancer. Materials and Methods: A total of 549 patients who had undergone surgery for gastric cancer at Osaka-City University were enrolled. LOX and LOXL2 expressions were evaluated by intensity of staining and percentage of stained tumor cells at the invading tumor front. This study was approved by the Osaka City University ethics committee. Informed consent was obtained from all patients. Results: LOX and LOXL2 expression were positive in 265 (48.3%) and 342 (58.5%) of the 549 gastric tumors. Both LOX and LOXL2 expression in cancer cells were significantly associated with macroscopic type (p<0.01), histological type (p<0.01), depth of invasion (p<0.01), lymph node status (p<0.01), vessel invasion (p<0.01). The prognosis of patients with LOX-positive tumors or LOXL2-positive tumors was significantly (p<0.001 or p<0.001, log-rank) worse than that of patients with LOX-negative or LOXL2-negative tumors, respectively. A multivariate analysis indicated LOX and LOXL2 expression in cancer cells to be an independent prognostic factor (p=0.016 or p<0.001). Conclusions: LOX and LOXL2 expression might be associated with metastatic activity of gastric cancer cells. LOX and LOXL2 might be useful predictive prognostic factors for patients with gastric cancer. Citation Format: Hiroaki Kasashima, Masakazu Yashiro, Yukie Go, Go Masuda, Haruhito Kinoshita, Mao Tokumoto, Tamami Morisaki, Tatsunari Fukuoka, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Role of lysyl oxidase and lysyl oxidase-like 2 in gastric carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2014-1980