LTF ameliorates cartilage endplate degeneration by suppressing calcification, senescence and matrix degradation through the JAK2/STAT3 pathway.

Intervertebral disc degeneration (IDD)-induced cervical and lumbar herniations are debilitating diseases. The function of intervertebral disc (IVD) mainly depends on the cartilage endplate (CEP), which provides support and waste removal. Therefore, IDD stems from the degeneration of CEP. Our study shows that the expression of lactotransferrin (LTF), an iron-binding protein, is significantly decreased in degenerated human and rat CEP tissues. In addition, we found that LTF knockdown promoted calcification, senescence, and extracellular matrix (ECM) degradation in human endplate chondrocytes. Furthermore, the in vivo experiment results confirmed that the JAK2/STAT3 pathway inhibitor AG490 significantly reversed these effects. In addition to investigating the role and mechanism of LTF in CEP degeneration, this study provides a theoretical basis and experimental evidence to improve IDD treatment.

CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway.

BACKGROUND: Osteoarthritis (OA), the most common joint disease, is linked with chondrocyte apoptosis and extracellular matrix (ECM) degradation. Charged multivesicular body protein 5 (CHMP5), a member of the multivesicular body, has been reported to serve as an anti-apoptotic protein to participate in leukemia development. However, the effects of CHMP5 on apoptosis and ECM degradation in OA remain unclear. METHODS: In this study, quantitative proteomics was performed to analyze differential proteins between normal and OA patient articular cartilages. The OA mouse model was constructed by the destabilization of the medial meniscus (DMM). In vitro, interleukin-1 beta (IL-1ß) was used to induce OA in human chondrocytes. CHMP5 overexpression and silencing vectors were created using an adenovirus system. The effects of CHMP5 on IL-1ß-induced chondrocyte apoptosis were investigated by CCK-8, flow cytometry, and western blot. The effects on ECM degradation were examined by western blot and immunofluorescence. The potential mechanism was explored by western blot and Co-IP assays. RESULTS: Downregulated CHMP5 was identified by proteomics in OA patient cartilages, which was verified in human and mouse articular cartilages. CHMP5 overexpression repressed cell apoptosis and ECM degradation in OA chondrocytes. However, silencing CHMP5 exacerbated OA chondrocyte apoptosis and ECM degradation. Furthermore, we found that the protective effect of CHMP5 against OA was involved in nuclear factor kappa B (NF-κB) signaling pathway. CONCLUSIONS: This study demonstrated that CHMP5 repressed IL-1ß-induced chondrocyte apoptosis and ECM degradation and blocked NF-κB activation. It was shown that CHMP5 might be a novel potential therapeutic target for OA in the future.

BASP1 knockdown suppresses chondrocyte apoptosis and extracellular matrix degradation in vivo and in vitro: A possible therapeutic approach for osteoarthritis.

Osteoarthritis(OA) is an age-related degenerative disease involving chondrocyte apoptosis and extracellular matrix(ECM) degradation.Brain acid soluble protein 1(BASP1) has been reported to induce apoptosis.Thus, we speculated that BASP1 might regulate OA progression by inducing apoptosis, which is also the purpose of this study.The cartilage of the knee joint was collected from OA patients who received the joint replacement.In OA cartilage tissue,we found BASP1 expression was highly expressed, which inferred that BASP1 might be involved in OA.To validate our hypothesis, destabilization of the medial meniscus (DMM) surgery-induced male C57BL/6mice and interleukin-1ß (IL-1ß)-treated human chondrocytes were used to mimic the OA environment.BASP1 knockdown in mice and chondrocytes was achieved by adenovirus carried with BASP1-specific shRNA.High expression of BASP1 was observed in OA mice, which was also verified in IL-1ß-treated chondrocytes.The potential mechanism of BASP1 in OA was further explored in vitro.BASP1 knockdown alleviated IL-1ß-induced apoptosis and ECM degradation, as reflected by the decreased number of apoptotic cells and matrix metalloproteases 13 expression,and the increased collagen II expression.Our findings indicated that BASP1 knockdown alleviated OA progression by inhibiting apoptosis and ECM degradation, suggesting that inhibiting BASP1 may be a potentially applicable method for preventing OA.

VEGF-D-mediated signaling in tendon cells is involved in degenerative processes.

Vascular endothelial growth factor (VEGF) signaling is crucial for a large variety of cellular processes, not only related to angiogenesis but also in nonvascular cell types. We have previously shown that controlling angiogenesis by reducing VEGF-A signaling positively affects tendon healing. We now hypothesize that VEGF signaling in non-endothelial cells may contribute to tendon pathologies. By immunohistochemistry we show that VEGFR1, VEGFR2, and VEGFR3 are expressed in murine and human tendon cells in vivo. In a rat Achilles tendon defect model we show that VEGFR1, VEGFR3, and VEGF-D expression are increased after injury. On cultured rat tendon cells we show that VEGF-D stimulates cell proliferation in a dose-dependent manner; the specific VEGFR3 inhibitor SAR131675 reduces cell proliferation and cell migration. Furthermore, activation of VEGFR2 and -3 in tendon-derived cells affects the expression of mRNAs encoding extracellular matrix and matrix remodeling proteins. Using explant model systems, we provide evidence, that VEGFR3 inhibition prevents biomechanical deterioration in rat tail tendon fascicles cultured without load and attenuates matrix damage if exposed to dynamic overload in a bioreactor system. Together, these results suggest a strong role of tendon cell VEGF signaling in mediation of degenerative processes. These findings give novel insight into tendon cell biology and may pave the way for novel treatment options for degenerative tendon diseases.

SNORC knockdown alleviates inflammation, autophagy defect and matrix degradation of chondrocytes in osteoarthritis development.

Excessive inflammation and autophagy defect of chondrocytes play important roles in the pathological process of osteoarthritis (OA). The present study aimed to clarify the roles of small novel rich in cartilage (SNORC) in these pathological changes of chondrocytes in OA. Bioinformatics analysis of GEO dataset GSE207881 displayed that SNORC was a potential biomarker for OA. As confirmed by quantitative real-time PCR, immunohistochemical staining and western blotting, SNORC was significantly up-regulated in cartilage of OA rat model and interleukin (IL)-1ß-stimulated primary rat articular chondrocytes in contrast to their corresponding normal control. Knocking down SNORC in IL-1ß-induced chondrocytes obviously suppressed the production of nitric oxide (NO), IL-6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) to alleviate inflammation, and reduced the protein levels of a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5) and matrix metallopeptidase (MMP)13 and elevated collagen type 2 alpha 1 (COL2A1) level to improve matrix degradation. Down-regulation of SNORC increased Beclin1 expression and LC3II/LC3I ratio, but suppressed p62 expression to restore impaired autophagy in IL-1ß-induced chondrocytes. Moreover, down-regulating SNORC mitigated mitochondrial dysfunction and apoptosis in IL-1ß-stimulated chondrocytes. Mechanically, SNORC simultaneously activated the phosphatidylinositol-3-kinase/serine threonine kinase (PI3K/AKT) and c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in the IL-1ß-induced chondrocyte, while re-activating the PI3K and JNK signals abolished the suppressive effect of down-regulating SNORC on IL-1ß-induced chondrocyte damage. In a word, SNORC knockdown alleviates inflammation, matrix degradation, autophagy defect and excessive apoptosis of chondrocytes during OA development via suppressing the PI3K and JNK signaling pathway.

Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1ß-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1.

Osteoarthritis (OA) is a form of chronic degenerative disease contributing to elevated disability rate among the elderly. Genkwanin is an active component extracted from Daphne genkwa possessing pharmacologic effects. Here, this study is designed to expound the specific role of genkwanin in OA and elaborate the probable downstream mechanism. First, the viability of chondrocytes in the presence or absence of interleukin-1 beta (IL-1ß) treatment was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to assess cell apoptosis. Inflammatory response was estimated through enzyme-linked immunosorbent assay and Western blot. In addition, immunofluorescence staining and Western blot were utilized to measure the expression of extracellular matrix (ECM)-associated proteins. Dual-specificity protein phosphatase-1 (DUSP1) expression was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Following DUSP1 elevation in genkwanin-treated chondrocytes exposed to IL-1ß, inflammatory response and ECM-associated factors were evaluated as forementioned. In addition, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining was to assess the mitochondrial membrane potential. Adenosine triphosphate (ATP) level was examined with ATP assay kit, and RT-qPCR was used to test mitochondrial DNA expression. Results indicated that genkwanin administration enhanced the viability while ameliorated the apoptosis, inflammatory response, and ECM degradation in IL-1ß-induced chondrocytes. Besides, genkwanin treatment fortified DUSP1 expression in IL-1ß-exposed chondrocytes. DUSP1 interference further offsets the impacts of genkwanin on the inflammation, ECM degradation, and mitochondrial dysfunction in IL-1ß-challenged chondrocytes. In short, genkwanin enhanced DUSP1 expression to mitigate mitochondrial dysfunction, thus ameliorating IL-1ß-elicited inflammation, apoptosis, and degradation of ECM in chondrocytes.

Embedding and Backscattered Scanning Electron Microscopy (EM-BSEM) Is Preferential over Immunophenotyping in Relation to Bioprosthetic Heart Valves.

Hitherto, calcified aortic valves (AVs) and failing bioprosthetic heart valves (BHVs) have been investigated by similar approaches, mostly limited to various immunostaining techniques. Having employed multiple immunostaining combinations, we demonstrated that AVs retain a well-defined cellular hierarchy even at severe stenosis, whilst BHVs were notable for the stochastic degradation of the extracellular matrix (ECM) and aggressive infiltration by ECM-digesting macrophages. Leukocytes (CD45+) comprised ≤10% cells in the AVs but were the predominant cell lineage in BHVs (≥80% cells). Albeit cells with uncertain immunophenotype were rarely encountered in the AVs (≤5% cells), they were commonly found in BHVs (≥80% cells). Whilst cell conversions in the AVs were limited to the endothelial-to-mesenchymal transition (represented by CD31+α-SMA+ cells) and the formation of endothelial-like (CD31+CD68+) cells at the AV surface, BHVs harboured numerous macrophages with a transitional phenotype, mostly CD45+CD31+, CD45+α-SMA+, and CD68+α-SMA+. In contrast to immunostaining, which was unable to predict cell function in the BHVs, our whole-specimen, nondestructive electron microscopy approach (EM-BSEM) was able to distinguish between quiescent and matrix-degrading macrophages, foam cells, and multinucleated giant cells to conduct the ultrastructural analysis of organelles and the ECM, and to preserve tissue integrity. Hence, we suggest EM-BSEM as a technique of choice for studying the cellular landscape of BHVs.

Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness.

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity.

HIF-1α aggravates pathologic myopia through the miR-150-5p/LAMA4/p38 MAPK signaling axis.

Therapeutic inhibition of hypoxia-inducible factor-1alpha (HIF-1α) action has emerged as a potential approach for managing several diseases, including myopia. Herein, we analyzed the role of HIF-1α in the progression of pathologic myopia by regulating the miR-150-5p/LAMA4/p38 MAPK axis. Microarray-based gene expression profiling of pathologic myopia was employed to identify differentially expressed genes. Human scleral fibroblasts (HSFs) were cultured under the hypoxic conditions. Interaction among HIF-1α, miR-150-5p, and LAMA4 was identified. Gain- and loss-of-function experiments were performed in hypoxia-exposed HSFs to evaluate the effect of the HIF-1α/miR-150-5p/LAMA4/p38 MAPK axis on the extracellular matrix (ECM) degradation of HSFs and the subsequent pathologic myopia progression. Increased LAMA4 but decreased miR-150-5p was found in serum sample of pathologic myopia patients. HIF-1α and LAMA4 were abundantly expressed, and p38 MAPK was activated while miR-150-5p was weakly expressed in hypoxia-exposed HSFs. HIF-1α was enriched in the promoter region of miR-150-5p and downregulated its expression, thus repressing the ECM degradation of HSFs as shown by increased COL1A1 and TIMP-2 and reduced MMP2. In addition, LAMA4 was a downstream target of miR-150-5p and under the negative regulation by miR-150-5p. Overexpression of miR-150-5p promoted the ECM degradation of HSFs by inhibiting LAMA4 expression and p38 MAPK signaling pathway. However, upregulation of LAMA4 reversed the promoting effect of miR-150-5p on ECM degradation of HSFs. Overall, our findings suggest that HIF-1α can decline miR-150-5p expression and facilitate LAMA4-mediated p38 MAPK signaling pathway activation, thus arresting ECM degradation of HSFs and eventually inducing pathologic myopia.

Extracellular matrix modulating enzyme functionalized biomimetic Au nanoplatform-mediated enhanced tumor penetration and synergistic antitumor therapy for pancreatic cancer.

BACKGROUND: Excessive extracellular matrix (ECM) deposition in pancreatic ductal adenocarcinoma (PDAC) severely limits therapeutic drug penetration into tumors and is associated with poor prognosis. Collagen is the most abundant matrix protein in the tumor ECM, which is the main obstacle that severely hinders the diffusion of chemotherapeutic drugs or nanomedicines. METHODS: We designed a collagenase-functionalized biomimetic drug-loaded Au nanoplatform that combined ECM degradation, active targeting, immune evasion, near-infrared (NIR) light-triggered drug release, and synergistic antitumor therapy and diagnosis into one nanoplatform. PDAC tumor cell membranes were extracted and coated onto doxorubicin (Dox)-loaded Au nanocages, and then collagenase was added to functionalize the cell membrane through lipid insertion. We evaluated the physicochemical properties, in vitro and in vivo targeting, penetration and therapeutic efficacy of the nanoplatform. RESULTS: Upon intravenous injection, this nanoplatform efficiently targeted the tumor through the homologous targeting properties of the coated cell membrane. During penetration into the tumor tissue, the dense ECM in the PDAC tissues was gradually degraded by collagenase, leading to a looser ECM structure and deep penetration within the tumor parenchyma. Under NIR irradiation, both photothermal and photodynamic effects were produced and the encapsulated chemotherapeutic drugs were released effectively, exerting a strong synergistic antitumor effect. Moreover, this nanoplatform has X-ray attenuation properties that could serve to guide and monitor treatment by CT imaging. CONCLUSION: This work presented a unique and facile yet effective strategy to modulate ECM components in PDAC, enhance tumor penetration and tumor-killing effects and provide therapeutic guidance and monitoring.

Paeonol inhibits inflammatory response and protects chondrocytes by upregulating sirtuin 1.

Paeonol is the bioactive component in Paeonia lactiflora Pall., Cynanchum paniculatum and Paeonia × suffruticosa Andr. Paeonol has been previously demonstrated to inhibit the release of tumor necrosis factor α (TNF-α) and interluekin 6 (IL-6) in chondrocytes. Sirtuin 1 (SIRT1) is downregulated in degraded cartilage and paeonol could induce nuclear accumulation of SIRT1. Therefore, the present study aims to investigate the possible role of paeonol in chondrocyte inflammation and cartilage protection in osteoarthritis (OA) as well as its regulation of SIRT1. Primary chondrocytes from rat knee joints were transfected with short hairpin (sh) - SIRT1 and (or) paeonol prior to IL-1ß exposure, and then inflammatory response, apoptosis, and extracellular matrix (ECM) degradation in the cells were evaluated concurrent with the activation of the nuclear factor κß (NF-κß) signaling pathway. Increased levels of TNF-α, IL-17, IL-6, matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 along with decreased tissue inhibitor of metalloproteinases 1 and type II collagen levels were found in IL-1ß-stimulated chondrocytes. Chondrocyte apoptosis was elevated and the NF-κß signaling pathway was activated in response to IL-1ß treatment. Paeonol enhanced SIRT1 expression to inactivate the NF-κß signaling pathway, thereby ameliorating inflammatory cytokine secretion, ECM degradation, and chondrocyte apoptosis. In conclusion, the results of the present study confirm the potential of paeonol as a candidate OA drug.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8.

BACKGROUND: Our previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear. METHODS: We examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague-Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury. RESULTS: MiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, ß-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats. CONCLUSIONS: Our data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness.

BACKGROUND: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation on its residue Ser5 is known to increase its actin-bundling activity. We recently showed that activation of the ERK/MAPK signalling pathway leads to L-plastin Ser5 phosphorylation and that the downstream kinases RSK1 and RSK2 are able to directly phosphorylate Ser5. Here we investigate the involvement of the PI3K pathway in L-plastin Ser5 phosphorylation and the functional effect of this phosphorylation event in breast cancer cells. METHODS: To unravel the signal transduction network upstream of L-plastin Ser5 phosphorylation, we performed computational modelling based on immunoblot analysis data, followed by experimental validation through inhibition/overexpression studies and in vitro kinase assays. To assess the functional impact of L-plastin expression/Ser5 phosphorylation in breast cancer cells, we either silenced L-plastin in cell lines initially expressing endogenous L-plastin or neoexpressed L-plastin wild type and phosphovariants in cell lines devoid of endogenous L-plastin. The established cell lines were used for cell biology experiments and confocal microscopy analysis. RESULTS: Our modelling approach revealed that, in addition to the ERK/MAPK pathway and depending on the cellular context, the PI3K pathway contributes to L-plastin Ser5 phosphorylation through its downstream kinase SGK3. The results of the transwell invasion/migration assays showed that shRNA-mediated knockdown of L-plastin in BT-20 or HCC38 cells significantly reduced cell invasion, whereas stable expression of the phosphomimetic L-plastin Ser5Glu variant led to increased migration and invasion of BT-549 and MDA-MB-231 cells. Finally, confocal image analysis combined with zymography experiments and gelatin degradation assays provided evidence that L-plastin Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity and concomitant extracellular matrix degradation. CONCLUSION: Altogether, our results demonstrate that L-plastin Ser5 phosphorylation increases breast cancer cell invasiveness. Being a downstream molecule of both ERK/MAPK and PI3K/SGK pathways, L-plastin is proposed here as a potential target for therapeutic approaches that are aimed at blocking dysregulated signalling outcome of both pathways and, thus, at impairing cancer cell invasion and metastasis formation. Video abstract.

Overexpression of long non-coding RNA XIST promotes IL-1ß-induced degeneration of nucleus pulposus cells through targeting miR-499a-5p.

BACKGROUND: Long non-coding RNA X-interactive specific transcript (XIST) is implicated in many diseases. However, its role and interaction with microRNA (miR)-499a-5p in intervertebral disc degeneration (IDD) remained unclear. METHODS: Nucleus pulposus (NP) tissue samples were collected and nucleus pulposus cells (NPCs) were isolated for Interleukin-1ß (IL-1ß) treatment and identification. XIST and miR-499a-5p expressions in the tissue were measured with quantitative real-time polymerase chain reaction (qRT-PCR). After IL-1ß treatment, NPC apoptosis was detected by flow cytometry. The potential binding sites of XIST and miR-499a-5p were predicted by starBase and confirmed by dual-luciferase reporter assay. Relative expressions of tissue inhibitor of metalloproteinases-3 (TIMP-3), Matrix metalloproteinases-3 (MMP-3), MMP-13, Collagen II, Aggrecan and apoptosis-related proteins (Bcl-2 associated X protein, Bax; B-cell lymphoma 2, Bcl-2; cleaved caspase-3) were measured by qRT-PCR and Western blot as needed. RESULTS: XIST expression was upregulated in the NP tissues of patients with IDD, and IL-1ß treatment resulted in a degradation of NPCs. Overexpressed XIST promoted the effects of IL-1ß on increasing NPC apoptosis and expressions of XIST, MMP-3, MMP-13, Bax and Cleaved caspase-3, but down-regulated TIMP-3, Collagen II, Aggrecan and Bcl-2 expressions. Silencing XIST, however, showed the opposite effects to its overexpression. MiR-499a-5p expression was downregulated in NP tissues of IDD patients and could bind with XIST, while its upregulation reversed the effects of overexpressed XIST in the IL-1ß-treated NPCs. CONCLUSION: Overexpressed XIST caused NPC degeneration through promoting apoptosis and extracellular matrix degradation of IL-1ß-treated NPCs through targeting miR-499a-5p, and therefore can serve as a potential treatment for IDD.

In vivo confocal Raman spectroscopic imaging of the human skin extracellular matrix degradation due to accumulated intrinsic and extrinsic aging.

BACKGROUND: Skin aging is a dynamic process that affects the entire body, marked by molecular and structural changes. Type I collagen is the most abundant structural component and accounts 80% of total collagen in human skin. The amount of proline and hydroxyproline reflect the quantity and quality of the collagen fiber in the extracellular matrix of skin, which is alerted due to accumulated effects of intrinsic and extrinsic aging. Extrinsic aging is driven by ultraviolet radiation-induced reactive oxygen species production that activates the matrix metalloproteinase and disrupts the extracellular matrix of skin dermis, while intrinsic aging is the non-enzymatic process resulting in advanced glycation end products (AGEs). In the presence of pentosidine-AGEs, aging process is accelerated. METHOD: In vivo Raman spectra of human dermis were collected from forearms of 30 volunteers and were divided into three groups: 10 young adult 25 ± 5 years, 10 old adult 65 ± 10 years and 10 diabetic old adult 65 ± 10 years old male participants. Density functional theory was performed to compute the vibration modes of AGEs, pentosidine, and glucosepane. RESULTS: In vivo confocal Raman spectroscopy detects the specific changes in the proline and hydroxyproline conformation, collagen fiber degradation of type I collagen and AGE protein contribution to specific Raman bands in the aged dermis because of Intrinsic and Extrinsic aging. Statistical t test marked significant differences (P < .01) in Raman peaks of proline and hydroxyproline among young adult, old adult, and diabetic old adult participants at wavenumbers 855, 875, 922, and 938 cm-1 . CONCLUSION: In vivo confocal Raman spectroscopy is a useful tool to detect the AGE markers in the old adult and diabetic old adult male participants, which interacts with the ultraviolet radiations and accelerates the aging process resulting in the extracellular matrix degradation.

MT1-MMP targeting to endolysosomes is mediated by upregulation of flotillins.

Tumor cell invasion and metastasis formation are the major cause of death in cancer patients. These processes rely on extracellular matrix (ECM) degradation mediated by organelles termed invadopodia, to which the transmembrane matrix metalloproteinase MT1-MMP (also known as MMP14) is delivered from its reservoir, the RAB7-containing endolysosomes. How MT1-MMP is targeted to endolysosomes remains to be elucidated. Flotillin-1 and -2 are upregulated in many invasive cancers. Here, we show that flotillin upregulation triggers a general mechanism, common to carcinoma and sarcoma, which promotes RAB5-dependent MT1-MMP endocytosis and its delivery to RAB7-positive endolysosomal reservoirs. Conversely, flotillin knockdown in invasive cancer cells greatly reduces MT1-MMP accumulation in endolysosomes, its subsequent exocytosis at invadopodia, ECM degradation and cell invasion. Our results demonstrate that flotillin upregulation is necessary and sufficient to promote epithelial and mesenchymal cancer cell invasion and ECM degradation by controlling MT1-MMP endocytosis and delivery to the endolysosomal recycling compartment.

MicroRNA-377-3p alleviates IL-1ß-caused chondrocyte apoptosis and cartilage degradation in osteoarthritis in part by downregulating ITGA6.

Increasing evidence indicates that altered expression of microRNAs (miRNAs) is associated with osteoarthritis (OA) progression. In our study, we demonstrated that miR-377-3p is underexpressed in OA-affected cartilage and IL-1ß-treated chondrocytes. Overexpression of miR-377-3p enhanced chondrocyte proliferation and restrained apoptosis and signs of cartilage matrix degradation and of an inflammatory response. Furthermore, ITGA6 was identified as a target gene of miR-377-3p. The latter was found to directly bind to the 3' untranslated region (3'UTR) of ITGA6 mRNA and downregulate ITGA6. In addition, ITGA6 expression was high in OA-affected tissues and negatively correlated with miR-77-3p expression. Overexpression of ITGA6 reversed the effects of miR-377-3p on IL-1ß-caused chondrocyte apoptosis, cartilage matrix degradation, and the inflammatory response. Moreover, bioinformatic analysis and a luciferase assay indicated that miR-377-3p expression is regulated by long noncoding RNA NEAT1, which binds to miR-377-3p and inactivates it. We showed that NEAT1 was highly expressed in OA-affected cartilage, negatively correlated with miR-377-3p levels, and positively correlated with ITGA6 levels. These findings provide information for the development of future treatments of OA, suggesting that miR-377-3p may be a therapeutic target in OA.

MiR-19b-3p attenuates IL-1ß induced extracellular matrix degradation and inflammatory injury in chondrocytes by targeting GRK6.

Osteoarthritis (OA) is characterized by degradation of articular cartilage. MiRNAs are involved in the regulation of chondrogenesis and OA. We aimed to investigate effects and mechanisms of miR-19b-3p in regulating chondrocytes viability, cartilage degradation and inflammatory response. Primary chondrocytes were isolated from cartilages in control subjects and patients with OA. Murine ATDC5 cells were pre-conditioned with IL-1ß in vitro. Expressions and interaction of miR-19b-3p with G protein-coupled receptor kinase 6 (GRK6), and their effects on inflammation, chondrocytes viability and cartilage degradation were determined after miR-19b-3p mimic or GRK6 siRNA transfection. MiR-19b-3p was significantly decreased in OA chondrocytes and IL-1ß-stimulated ATDC5 cells, in paralleled with the elevated type-II-collagen, aggrecan, MMP13 and GRK6 expression. MiR-19b-3p mimic dramatically increased the viability of chondrocytes and suppressed cell apoptosis. It also increased type-II-collagen, aggrecan expression and glycosaminoglycan (sGAG) content, and decreased the expression of MMP-1 and MMP-13 that controlled by IL-1ß. Overexpression of miR-19b-3p inhibited the production of IL-6 and IL-8 in ATDC5 cells. However, the protective effects of miR-19b-3p mimic on IL-1ß induced cell death; IL-8 production and sGAG decrease were greatly discounted by GRK6 lentiviral vectors. Luciferase reporter assay confirmed that GRK6 gene was a direct target ofmiR-19b-3p. GRK6 siRNA transfection antagonized the IL-1ß-induced chondrocytes injury, extracellular matrix degradation and inflammatory response. MiR-19b-3p mimic and GRK6 siRNA showed comparable inhibitory effect on IL-1ß-provoked NF-κB as reflected by the expression of p-p65. NF-κB translocation inhibition with PS1154 reversed the effects of IL-1ß on IL-8 and sGAG. Collectively, miR-19b-3p attenuated OA by targeting GRK6-NF-κB pathway.

Study on the tumor-induced angiogenesis using mathematical models.

We studied angiogenesis using mathematical models describing the dynamics of tip cells. We reviewed the basic ideas of angiogenesis models and its numerical simulation technique to produce realistic computer graphics images of sprouting angiogenesis. We examined the classical model of Anderson-Chaplain using fundamental concepts of mass transport and chemical reaction with ECM degradation included. We then constructed two types of numerical schemes, model-faithful and model-driven ones, where new techniques of numerical simulation are introduced, such as transient probability, particle velocity, and Boolean variables.

Alpha-1 Antitrypsin-Deficient Macrophages Have Increased Matriptase-Mediated Proteolytic Activity.

Alpha-1 antitrypsin (AAT) deficiency-associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (Mɸ) express AAT, we propose that the expression and intracellular accumulation of mutated Z-AAT (the most common mutation) compromises Mɸ function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, Mɸ from individuals with Z-AAT (Z-Mɸ) have greater proteolytic activity on ECM than do normal Mɸ. This abnormal Z-Mɸ activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z-AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z-Mɸ. Importantly, compared with normal Mɸ, the membrane-bound serine protease, matriptase, is present in Z-Mɸ at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)-14, a membrane-anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP-14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z-Mɸ, both directly and through MMP-14 activation. In summary, the expression of Z-AAT in Mɸ confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency-associated emphysema.