Increased Collagen Degradation Research Articles

Cell Senescence-Independent Changes of Human Skin Fibroblasts with Age.

Skin ageing is defined, in part, by collagen depletion and fragmentation that leads to a loss of mechanical tension. This is currently believed to reflect, in part, the accumulation of senescent cells. We compared the expression of genes and proteins for components of the extracellular matrix (ECM) as well as their regulators and found that in vitro senescent cells produced more matrix metalloproteinases (MMPs) than proliferating cells from adult and neonatal donors. This was consistent with previous reports of senescent cells contributing to increased matrix degradation with age; however, cells from adult donors proved significantly less capable of producing new collagen than neonatal or senescent cells, and they showed significantly lower myofibroblast activation as determined by the marker α-SMA. Functionally, adult cells also showed slower migration than neonatal cells. We concluded that the increased collagen degradation of aged fibroblasts might reflect senescence, the reduced collagen production likely reflects senescence-independent processes.

Rapamycin Insensitive Regulation of Engineered Ligament Structure and Function by IGF-1.

Following rupture, the anterior cruciate ligament (ACL) will not heal and therefore more than 400,000 surgical repairs are performed annually. Ligament engineering is one way to meet the increasing need for donor tissue to replace the native ligament; however, currently these tissues are too weak for this purpose. Treating engineered human ligaments with insulin-like growth factor-1 (IGF-1) improves the structure and function of these grafts. Since the anabolic effects of IGF-1 are largely mediated by mTORC1, we used rapamycin to determine whether mTORC1 was necessary for the improvement in collagen content and mechanics of engineered ligaments. The effect of IGF-1 and rapamycin were determined independently and interactions between the two treatments were tested. Grafts were treated for 6 days before mechanical testing and analysis of collagen content. Following 8 days of treatment, mechanical properties increased 34% with IGF-1 and decreased 24.5% with rapamycin. Similarly, collagen content increased 63% with IGF-1 and decreased 36% with rapamycin. Interestingly, there was no interaction between IGF-1 and rapamycin, suggesting that IGF-1 was working in a largely mTORC1-independent manner. Acute treatment with IGF-1 did not alter procollagen synthesis in growth media, even though rapamycin decreased procollagen 55%. IGF-1 decreased collagen degradation 15%, whereas rapamycin increased collagen degradation 10%. Once again, there was no interaction between IGF-1 and rapamycin on collagen degradation. Together, these data suggest that growth factor-dependent increases in collagen synthesis are dependent on mTORC1 activity; however, IGF-1 improves human engineered ligament mechanics and collagen content by decreasing collagen degradation in a rapamycin-independent manner.

Matrix Metalloproteases 8 Polymorphism as Risk Factor for Rotator Cuff Tear.

Rotator Cuff Tear (RCT) is a multifactorial disease, but an important one is the increased collagen degradation that would lead to a higher chance of tear. MMP-8 is a protein that degrades type I collagen, and it is known that MMP-8 has a polymorphism in which a T allele in the gene promoter region increases its transcription activity. This study aims to investigate the association between MMP-8 polymorphism g.-799 C>T (rs11225394) and RCT. To do that, we collected DNA samples from buccal epithelial cells of 128 patients (separated into RCT group and control group in a proportion 1:1) and genotyped the DNA using PCR. The statistical analyses were done using the ARLEQUIN Version 2.0, and the data normality was tested with the Shapiro-Wilk test. The results showed a significantly higher frequency of T/T genotype in the test group (29% in the control group and 39% in the test group, p=0.0417), and that would represent a risk factor for increased collagen degradation. The MMP-8 g.-799 C>T (rs11225394) SNP was associated with RCT. With the description of a new risk factor, future research can be done to analyze how to prevent RCT or develop new treatment strategies since the disease's failure index is currently high.

17α-estradiol, a lifespan-extending compound, attenuates liver fibrosis by modulating collagen turnover rates in male mice.

Estrogen signaling is protective against chronic liver diseases, although men and a subset of women are contraindicated for chronic treatment with 17β-estradiol (17β-E2) or combination hormone replacement therapies. We sought to determine if 17α-estradiol (17α-E2), a naturally occurring diastereomer of 17β-E2, could attenuate liver fibrosis. We evaluated the effects of 17α-E2 treatment on collagen synthesis and degradation rates using tracer-based labeling approaches in male mice subjected to carbon tetrachloride (CCl4)-induced liver fibrosis. We also assessed the effects of 17α-E2 on markers of hepatic stellate cell (HSC) activation, collagen cross-linking, collagen degradation, and liver macrophage content and polarity. We found that 17α-E2 significantly reduced collagen synthesis rates and increased collagen degradation rates, which was mirrored by declines in transforming growth factor β1 (TGF-β1) and lysyl oxidase-like 2 (LOXL2) protein content in liver. These improvements were associated with increased matrix metalloproteinase 2 (MMP2) activity and suppressed stearoyl-coenzyme A desaturase 1 (SCD1) protein levels, the latter of which has been linked to the resolution of liver fibrosis. We also found that 17α-E2 increased liver fetuin-A protein, a strong inhibitor of TGF-β1 signaling, and reduced proinflammatory macrophage activation and cytokines expression in the liver. We conclude that 17α-E2 reduces fibrotic burden by suppressing HSC activation and enhancing collagen degradation mechanisms. Future studies will be needed to determine if 17α-E2 acts directly in hepatocytes, HSCs, and/or immune cells to elicit these benefits.

Melatonin attenuates thioacetamide-induced liver fibrosis in male rats through modulation of interleukin-6, interleukin-4, apoptosis and urokinase plasminogen activator receptor-associated protein/Endo180.

Liver fibrosis is a chronic progressive disease, its resolution still unclear, and the current study explored the role of melatonin in modulation of interleukin-6 (IL-6), interleukin-4 (IL-4), transforming growth factor beta1 (TGF-β1) and urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) pathway in thioacetamide (TAA)-induced hepatotoxicity. Thirty two adult Sprague-Dawley rats were divided into four groups: vehicle control group, TAA-induced liver fibrosis group that was left untreated, melatonin administration before and along with TAA and melatonin along with TAA group. TTA-induced massive liver necrosis, fibrosis around portal tract and increases serum levels of liver enzymes and total bilirubin when compared with control vehicle group. While both melatonin pretreatment and treatment retained liver parenchyma and liver enzymes quite similar to control group and reduced TAA-induced liver injury. Notably, melatonin pretreatment and treatment increased collagen degradation in TAA liver injury by19, 31.7-fold respectively evidence by collagen percentage area. Melatonin also decreased the amount of thiobarbituric acid reactive compounds and retained the reduced glutathione and superoxide dismutase to basal level quite similar to control group. Additionally, melatonin significantly (P value ≤0.05) decreased the levels of TGF-β1, epidermal growth factor (EGF), hydroxyproline, tissues IL-6, caspase-3, and receptor interacting serine/threonine kinase1 (RIPK1), fibrillin-1, and - smooth muscle actin in the liver tissues while significantly (P value ≤0.05) increasing the levels of IL-4 and uPARAP/Endo180. Due to its anti-inflammatory, anti-apoptotic, and antioxidant capabilities as well as its ability to decrease hepatic stellate cell activation and fibrogenesis, these data imply that melatonin has a powerful anti-fibrotic effect.

Anti-Aging Effect and Mechanism of Proanthocyanidins Extracted from Sea buckthorn on Hydrogen Peroxide-Induced Aging Human Skin Fibroblasts.

Oxidative stress is the leading cause of skin aging damage. Excessive accumulation of reactive oxygen species (ROS) in cells induced by hydrogen peroxide (H2O2) triggers a decrease in collagen synthesis and an increase in collagen degradation, which are biomarkers of skin aging. We evaluated the potential protective mechanism of Sea buckthorn proanthocyanidins (SBP) against the oxidative stress-induced skin aging process from multiple aspects. We treated human skin fibroblasts (HSFs) with 300 µmoL/L of H2O2 for 24 h, followed by 25, 50, and 100 µg/mL of SBP for 24 h. The results showed that SBP could enhance the activities of superoxide dismutase (SOD) and glutathione (GSH), effectively remove excess ROS, and significantly improve the changes in cell morphology and viability caused by excessive ROS in skin cells. In addition, SBP could promote the synthesis of Col I in aging HSFs through the TGF-β1/Smads pathway and inhibit the degradation of Col I by regulating the MMPs/TIMPs system, thereby maintaining the stability of the ECM structure to achieve anti-aging purposes. Finally, we studied the migration ability of SBP, and the results showed that 100 µg/mL of SBP was most conducive to the cell migration of senescent cells, laying a foundation for follow-up animal experiments. These results will increase the application value of SBP in the cosmetic and antioxidative functional food industries.

Overexpression of V-ATPase B2 attenuates lung injury/fibrosis by stabilizing lysosomal membrane permeabilization and increasing collagen degradation

Excessive oxidative stress causes lysosomal membrane permeabilization (LMP), which leads to cell death. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H+ into the cytosol and thus maintaining intracellular pH. Previously, we reported that V-ATPase B2 subunit expression is upregulated in the TiO2-exposed lung epithelium. We investigated the role of the lysosomal V-ATPase B2 subunit in oxidative stress-induced alveolar epithelial cell death and in an experimental lung injury/fibrosis model. Overexpression of V-ATPase B2 increased lysosomal pH and lysosomal activities in the cells. In the presence of H2O2, overexpression of V-ATPase B2 increased survival, and silencing of V-ATPase B2 dramatically increased cell death. Overexpression of V-ATPase B2 diminished H2O2-triggered LMP, as evidenced by a reduction in acridine orange staining and leakage of cathepsin D from the lysosome to the cytoplasm. In addition, V-ATPase B2-overexpressing macrophages exhibited significantly enhanced uptake and degradation of collagen. V-ATPase B2-overexpressing transgenic mice showed significant inhibition of the bleomycin-induced increases in lung inflammation and fibrosis. We conclude that V-ATPase B2 is critical for maintaining lysosomal activities against excessive oxidative stress by stabilizing LMP. Our findings reveal a previously unknown role of this V-ATPase subunit in a lung injury and fibrosis model.

Changes in Serum Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinases-1 in Patients with Essential Hypertension.

Hypertension (HTN) is a leading risk factor for cardiovascular (CV) disease. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are thought to be actively involved in the remodeling of the CV extracellular matrix (ECM) during hypertensive damage. Therefore, in this study, we aimed to investigate serum levels of MMP-1 and TIMP-1 in patients with essential HTN and compare them with those of normotensive individuals. We measured serum concentrations of MMP-1 and TIMP-1 in 60 patients with HTN and 20 healthy controls using an ELISA. The obtained results showed that in patients with HTN, the mean levels of MMP-1 (1.82 ± 0.9 ng/mL) were significantly higher (p = 0.03) than the mean levels in the control group (1.19 ± 0.7 ng/mL). The levels of TIMP-1 in patients with essential HTN (0.44 ± 0.1 ng/mL) were also significantly higher (p = 0.005) than those in the control group (0.33 ± 0.1 ng/mL). In HTN, elevated serum MMP-1 levels may be associated with increased collagen degradation in the CV ECM, whereas elevated TIMP-1 levels may favor its accumulation and the development of pathological remodeling and fibrosis of the heart and arterial vessels.

Coordination of two kinesin superfamily motor proteins, KIF3A and KIF13A, is essential for pericellular matrix degradation by membrane-type 1 matrix metalloproteinase (MT1-MMP) in cancer cells

MT1-MMP plays a crucial role in promoting the cellular invasion of cancer cells by degrading the extracellular matrix to create a path for migration. During this process, its localization at the leading edge of migrating cells is critical, and it is achieved by targeted transport of MT1-MMP-containing vesicles along microtubules by kinesin superfamily motor proteins (KIFs). Here we identified three KIFs involved in MT1-MMP vesicle transport: KIF3A, KIF13A, and KIF9. Knockdown of KIF3A and KIF13A effectively inhibited MT1-MMP-dependent collagen degradation and invasion, while knockdown of KIF9 increased collagen degradation and invasion. Our data suggest that KIF3A/KIF13A dependent MT1-MMP vesicles transport takes over upon KIF9 knockdown. Live-cell imaging analyses have indicated that KIF3A and KIF13A coordinate to transport the same MT1-MMP-containing vesicles from the trans-Golgi to the endosomes, and KIF13A alone transports the vesicle from the endosome to the plasma membrane. Taken together, we have identified a unique interplay between three KIFs to regulate leading edge localization of MT1-MMP and MT1-MMP-dependent cancer cell invasion.

The Correlation between Serum Cortisol Levels with Stretch Marks in Gymnastic Male

BACKGROUND: Stretch marks are skin scar tissue that appears in the form of purplish linear atrophy, erythematous or hypopigmented which is often caused by excessive stretching of the skin. Increased cortisol levels can cause an increase in collagen degradation which results in disruption of the extracellular matrix in the dermis, resulting in stretch marks. Physical stress can trigger activation of the hypothalamic-pituitary-adrenal axis, which will induce activation of stress hormones, including cortisol in the adrenal cortex. AIM: The objective of the study is to determine the correlation between serum cortisol levels and stretch marks in male at a gymnastics training site. SUBJECTS AND METHODS: Observational analytic study with a cross-sectional approach to 50 stretch marks subjects. RESULTS: Serum cortisol levels of subjects with stretch marks averaged 9.72 g/dL with the lowest level of 4.45 g/dL and the highest level of 49.25 g/dL (p < 0.001). The highest age with stretch marks was 26–30 years 18 (36%) subjects and the lowest age was aged 36–40 years 5 (10%) subjects. The majority of stretch marks are located in the axillary region (30.9%), brachii (23.6%), and abdomen (18.4%). The average cortisol level in subjects with aerobic exercise was 6.52 g/dL, muscle training 11.18 g/dL, mixed aerobic and muscle training 7.5 g/dL. The highest average cortisol levels were at exercise duration of 31–60 min of 12.88 g/dL, 61–90 min of 6.63 g/dL, and 91–120 min of 6.2 g/dL. The highest frequency of exercise in a week was 3–4 times as many as 30 subjects (60%) with an average serum cortisol level of 11.1879 g/dL. CONCLUSION: There is a significant correlation between serum cortisol levels and stretch marks in male at gymnastics training.

An Adipose-Derived Injectable Sustained-Release Collagen Scaffold of Adipokines Prepared Through a Fast Mechanical Processing Technique for Preventing Skin Photoaging in Mice.

Ultraviolet A (UVA) radiation is the major contributor to skin photoaging, associated with increased collagen degradation and reactive oxygen species (ROS) expression. Adipokines have been proven as promising therapeutic agents for skin photoaging. However, adipokine therapy is generally limited by the short in vivo release duration and biological instability. Therefore, developing a treatment that provides a sustained release of adipokines and enhanced therapeutic effects is desirable. In this study, we developed a novel mechanical processing technique to extract adipose tissue-derived ECM components, named the “adipose collagen fragment” (ACF). The physical characterization, injectability, collagen components, residual DNA/RNA and adipokine release pattern of ACF were identified in vitro. L929 cells were treated with ACF or phosphate-buffered saline for 24 h after UVA irradiation in vitro. The expression of senescence-associated xβ-galactosidase (SA-β-gal), ROS and antioxidase were investigated. Then, we evaluated its therapeutic efficacy by injecting ACF and phosphate-buffered saline, as a control, into the dermis of photoaging nude mice and harvesting skin samples at weeks 1, 2, and 4 after treatment for assessment. The content of adipokines released from ACF was identified in vivo. The collagen synthesis and collagen degradation in ACF implants were evaluated by immune staining. Dermal thickness, fibroblast expression, collagen synthesis, ROS level, antioxidase expression, capillary density, and apoptotic cell number were evaluated by histological assessment, immune staining, and polymerase chain reaction in the skin samples. We demonstrated that ACF is the concentrated adipose extracellular matrix collagen fragment without viable cells and can be injected through fine needles. The lower expression of SA-β-gal, ROS and higher expression of antioxidase were observed in the ACF-treated group. ACF undergoes collagen degradation and promotes neocollagen synthesis in ACF implants. Meanwhile, ACF serves as a sustained-release system of adipokines and exhibits a significantly higher therapeutic effect on mouse skin photoaging by enhancing angiogenesis, antioxidant abilities, antiapoptotic activities, and collagen synthesis through sustainedly releasing adipokines. To sum up, ACF is an adipokines-enriched, sustained-release extracellular matrix collagen scaffold that can prevent UVA-induced skin photoaging in mice. ACF may serve as a novel autologous skin filler for skin rejuvenation applications in the clinic.

Specific Parameters of Extracellular Matrix Remodeling of Liver and Lungs of Mice with BCG Granulomatosis during Chronic Inflammation Depending on the Method of Administration of Liposomal Oxidized Dextran

The objective: to study the effect of liposomal oxidized dextran (LOD) on remodeling of the extracellular matrix of organs of mice during the period of chronic BCG granulomatosis and effects of LOD on this process depending on the route of administration. Subjects and Methods. The liver and lungs of mice with BCG granulomatosis were studied using different methods of LOD administration (intraperitoneally and by inhalation). The content of glycosaminoglycans, hydroxyproline fraction, the activity of matrix metalloproteinases (MMPs), hyaluronidases and a2-macroglobulin, and the content of tissue inhibitors of MMPs (TIMP-1 and -2) were evaluated. Results. The administration of LOD to mice infected with the BCG vaccine suffering from chronic granulomatosis with severe fibrosis (6 months after infection for 3 months) resulted in the aggravation of collagen degradation in the liver. In the lungs, along with increased collagen degradation, decreased collagen synthesis was observed. It was apparently due to suppressed activity of a2-macroglobulin and decreased content of TIMP-1 and -2. In the liver, with intraperitoneal administration of LOD, signs of suppressed fibrogenesis and fibrolysis were observed versus the data obtained for inhalation administration. There were no differences in the content of hydroxyproline fractions in the lungs depending on the method of LOD administration. Thus, administration of LOD to mice led to lower severity of fibrosis while the mechanisms of fibrolysis in the lungs and liver differed.

Metformin Reduced Collagen Deposition and Contractility, but Increased Collagen Degradation in in vitro Posterior Capsule Opacification Model

BACKGROUND: Posterior capsule opacification (PCO) often occurs after cataract surgery. Metformin has been known to have an ability to inhibit fibrosis. This study aimed to investigate the effects of metformin on cell contractility, collagen deposition and degradation in human lens epithelial cells (HLEC) of cataract patients.METHODS: HLEC were isolated from the anterior lens capsule of patients undergoing cataract surgery. The HLEC culture was carried out using explant culture technique. The in vitro PCO model was created by scratching technique on HLEC cultures. The treatment groups were given 0.1, 0.5 and 1 mM metformin, respectively, while the control group were given 10% fetal bovine serum (FBS). On the 7th day after scratching technique, the collagen deposition, collagen degradation and cell contractility were evaluated.RESULTS: Collagen deposition in HLEC was significantly reduced after given 0.1 mM, 0.5 mM and 1 mM metformin (17.92±6.16 mg/mL, 12.92±4.31 mg/mL, 11.25±5.30 mg/mL, respectively), compared to the control group (31.46±7.52 μg/mL, p=0.002). Collagen degradation significantly was increased in the 0.1 mM, 0.5 mM and 1 mM metformin groups (4.77±9.27 mg/mL, 6.59±1.16 mg/mL, 6.35±1.90 mg/mL, respectively) compared to the control group (2.21±2.78 μg/mL, p=0.002). While, collagen contractility in HLEC was significantly reduced in 0.1mM, 0.5mM and 1 mM metformin groups (16.39±3.89%, 13.89±2.59%, 11.93±2.44%, respectively), compared to the control group (44.25±4.95%, p=0.000).CONCLUSION: Metformin reduced collagen deposition and contractility, but increased collagen degradation in HLEC of cataract patients through mechanism of extracellular matrix remodeling.KEYWORDS: metformin, human lens epithelial cell, fibrosis

Antioxidant and skin anti-aging effects of the aqueous ethanol extract of Ginkgo biloba leaf: an in vitro study using HaCaT keratinocytes

Oxidative stress is considered a prime contributor to skin aging. Aged skin can be characterized by increased wrinkles and dry and dull appearance, which are associated with increased collagen degradation and inadequate desquamation, respectively. We investigated the antioxidant and skin anti-aging effects of a Ginkgo biloba leaf extract (GLE), an aqueous ethanol extract of Ginkgo biloba leaves, and the molecular mechanisms involved, using HaCaT keratinocytes. HaCaT cells were pretreated with GLE (0–15 μg/mL) and then treated with H2O2 (0.2 mM), and the cell viability and metalloproteinase 1 (MMP-1) protein level were measured. To investigate the mechanisms underlying the GLE’s effects, the cells were incubated with GLE, and then glutathione content, cell migration rate, and the levels of proteins, i.e., MMPs, Kallikrein-related peptidases (KLKs), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and mitogen-activated protein kinases, were measured. GLE pretreatment caused decreases in cytotoxicity and the level of MMP-1, a key enzyme that degrades collagen in dermis, induced by H2O2 in HaCaT cells. GLE (5–15 μg/mL) caused increases in glutathione content and HO-1 level in cells, which appeared to be associated with the increase in Nrf2, a principal transcription factor for the antioxidant response. GLE also increased keratinocyte migration, which might result from the increase in KLK-7, a key enzyme that degrades corneodesmosomes and thus stimulates desquamation of corneocytes in epidermis. GLE increased the phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, which might contribute to the antioxidant and skin anti-aging effects of GLE demonstrated in our results. Our results suggest that GLE may have skin anti-aging effects by protecting keratinocytes against excessive oxidative stress via stimulation of antioxidant response, reducing wrinkle formation via a decrease in MMP-1 expression, and stimulating desquamation via an increase in KLK-7 expression.

Mannose-6-phosphate complex and improvement in biomechanical properties of the skin.

BackgroundThe dermis is composed of a tangle of macromolecules that provides the skin its biomechanical properties. During chronological aging, fibroblasts lose their ability to synthesize collagen and an accumulation of matrix metalloproteinases leads to an increase in collagen degradation. As a result, there is a decline in the biomechanical properties of the skin. Skin aging is accelerated by external factors such as UV radiation and pollution, which induce accumulation of oxidants, and so of oxidized proteins in the skin.AimsAtomic force microscopy (AFM) has emerged as an alternative method for studying the biomechanical properties of skin cells and tissues.Methods/ResultsThus, we identified mannose‐6‐phosphate complex as a new powerful molecule capable of reversing the visible signs of aging by reorganizing the collagen network of the dermis and by improving the skin biomechanical properties. This effect was correlated with clinical studies that showed a marked antiaging effect through a reduction in the number of crow's feet and in the depth and size of neck wrinkles.ConclusionMannose‐6‐phosphate complex appeared to be able to protect proteins in the dermis scaffold against oxidation and degradation, allowing an improvement in the skin biomechanical properties.

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 μg/mL bacterial collagenase at 37 °C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm-1. Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Low dietary silicon supplementation may not affect bone and cartilage in mature, sedentary horses

As osteoarthritis is a major cause of lameness in horses in the United States, improving collagen health prior to onset and increasing collagen turnover within affected joints could improve health- and welfare-related outcomes. Through its positive effects on bone mineral content and density and its role in increasing collagen synthesis, silicon (Si) may slow the development and progression of osteoarthritis, thereby reducing lameness. This study evaluated the hypothesis that Si supplementation would increase cartilage turnover through increased collagen degradation and formation markers, as well as bone formation markers, resulting in reduced lameness severity when compared with controls. Ten mature Standardbred geldings were assigned to either a Si-treated (SIL) or control (CON) group and group-housed on pasture for 84 d. Horses were individually fed to ensure no cross-contamination of Si other than what was present in the environment. For the duration of the study, SIL horses received a Si-collagen supplement at the rate of 0.3 g supplement/(100 kg body weight day). Serum samples were taken weekly for osteocalcin, and plasma samples were taken on days 0, 42, and 84 for plasma minerals. On days 0, 42, and 84, subjective and objective lameness exams were performed, and radiographs and synovial fluid samples were taken from reference and osteoarthritic joints. Plasma minerals were similar in both groups and were lower on day 84 than on day 0 (P < 0.05). Si supplementation, fed at the manufacturer's recommended rate, did not improve lameness or radiographs when compared with controls, and supplemented horses did not show greater collagen degradation and/or synthesis markers in synovial fluid than controls, indicating that cartilage turnover remained unaffected. However, a minimum beneficial threshold and range for Si supplementation standardized to body weight need to be established.

Matrix Metalloproteinase-3 is Key Effector of TNF-α-Induced Collagen Degradation in Skin.

Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (p < 0.001) in KO skin explants compared to WT control skin but did not differ (p = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (p = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.

554 Skin ageing continues long after ultraviolet radiation damage

Wrinkling has been attributed to the cumulative effects of acute Ultraviolet Radiation (UVR) exposure on the dermis. Previous studies have shown that the collagenase MMP-1 is secreted by dermal fibroblasts and degrades collagen immediately after UV exposure. However, skin ages gradually, and not intermittently after sun exposure. In this study we sought to examine the mechanism underpinning the gradual appearance of wrinkles, and to determine whether this is a result of intrinsic changes in the biology of the dermis. In vitro studies were performed using patient-derived human dermal fibroblasts from healthy skin, HFF and HDF cell lines. Cell lines were UV-protected or repeatedly exposed to UVB. Dermal fibroblasts from fairer skin types had higher levels of UV-induced mutations which, in multivariate analysis, was linked to higher expression of extracellular matrix degradation genes. This suggests that the background UVR exposure history influences the composition of the dermis as it ages. We investigated the mechanism driving collagen degradation in dermal fibroblasts with and without a history of UV damage. Fibroblasts with high background UV exposure have elevated levels of MMP-1 at rest. In addition, they show increased collagen degradation activity, which persists in the absence of acute UV exposure. Our study shows that dermal fibroblasts with a history of chronic UV exposure have higher levels of collagenase expression and activity, even in the absence of acute UV exposure. Importantly, these findings explain the mechanism driving gradual collagen degradation and wrinkling in the absence of UV light.

Abstract PD9-07: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with high risk breast cancer (BC): Role of collagen processing and tumor microenvironment

Abstract Background: Copper is an important catalytic cofactor in several biological functions and is essential for lysyl oxidase (LOX), a key enzyme in cross-linking collagen, which may play a role in tumor metastasis. We hypothesized that tetrathiomolybdate (TM)-associated copper depletion (CD) would inhibit tumor metastases by altering copper dependent collagen remodeling in the pre-metastatic niche. These results are an update of our previously reported clinical outcomes with longer follow-up and translational outcomes implicating the tumor microenvironment in metastatic transformation of BC. Methods: Pts at high risk for recurrence, node+ triple negative (TNBC) or stage 3/4 BC with no evidence of disease (NED), were enrolled on a phase II study of CD with TM. TM was given to maintain ceruloplasmin (Cp) levels between 8-16 mg/dl for two years (yrs) with an extension phase or until relapse. Median Cp levels were monitored with each cycle. Clinical endpoints included safety/tolerability and progression of disease (POD)/death. Event-free (EFS) and overall survival (OS) were calculated using Kaplan Meier survival analyses. Translational endpoints included markers of collagen cross-linking (LOXL-2), formation (PRO-C3), and degradation (C1M and C6M). Results: Seventy-five pts received 2993 cycles of TM on the primary (24 cycles, 28 days per cycle) and extension study. Median age was 51 yrs (range 29-66). Forty-five pts had stage 2/3 BC, and 30 pts were stage 4 NED. At a median follow-up of 8.4 yrs, the overall EFS was 71.4% and OS was 78.8%. The EFS and OS for the 36 pts with TNBC were 71.7% and 81%, and the EFS and OS for the 39 pts with Luminal/HER2+ BC were 71.2% and 78.6% respectively. TM was well tolerated with grade 3/4 toxicities including: neutropenia (1.9%), febrile neutropenia (0.03%), and fatigue (0.2%). LOXL2 levels were significantly decreased at 12 and 24 cycles compared with baseline (p&amp;lt;0.01) in those who were NED but not in those who had progressive disease (POD). LOXL2 levels were significantly correlated with C1M levels (spearman coefficient -0.34, p=0.02). C1M levels were significantly increased at 5, 11 and 24 cycles as compared with baseline (p&amp;lt;0.01) in those who were NED and were significantly higher as compared to levels in those experiencing POD/death, p&amp;lt;0.05. This difference may be more pronounced in those not achieving adequate CD (&amp;lt;50%) and in luminal/HER2+ BC. Interestingly, the ratio of C1M/PRO-C3 was significantly more elevated over time in those NED as compared to those experiencing POD/death. No associations were found with other collagen markers (PRO-C3 and C6M). Conclusions: TM is safe, well-tolerated and associated with decreased LOXL-2 and increased C1M levels over time in NED pts. This suggests that copper depletion may result in decreased collagen crosslinking and increased collagen degradation over formation, potentially “normalizing” the collagen microenvironment to create an inhospitable environment for tumor metastases. Larger randomized trials in high risk populations with translational outcomes are needed to further investigate the role of collagen processing in the tumor microenvironment and its potential as a biomarker of response. Citation Format: Liu YL, Bager CL, Willumsen N, Kornhauser N, Cobham M, Andreopoulou E, Cigler T, Moore A, LaPolla D, Fitzpatrick V, Ward M, Warren JD, Mittal V, Vahdat LT. A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with high risk breast cancer (BC): Role of collagen processing and tumor microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-07.