TIMP-1 Production Research Articles

(248) Analysis of Biomarkers (MMP-9, TIMP-1, Ki-67 and Type IV Collagen) in the Diagnosis of Prostate Diseases

Abstract Introduction The combination of inflammatory, benign and malignant processes in the prostate reduces the sensitivity, specificity and diagnostic accuracy of modern examination methods. The development of immunohistochemical methods (IHC) promotes the search for molecular markers that are diagnostically significant in various processes in the prostate. This will improve the detection of malignant neoplasms and reduce the number of "unnecessary" prostate biopsies. Objective to study in biopsies of patients with various prostate diseases the relationship of type 9 metalloproteinase (MMP-9) and its inhibitor (TIMP-1), the role of the Ki-67 protein proliferation level and the diagnostic value of the type IV collagen distribution indicator. Methods The study included 263 patients. They were divided into 4 groups: chronic prostatitis - 21 patients (mean age 65.2 ± 0.7 years), benign prostatic hyperplasia (BPH) - 65 patients (69.3 ± 1.1 years), BPH + prostatic intraepithelial neoplasia (PIN) - 59 patients (68.9 ± 0.9 years), prostate adenocarcinoma (PC) - 118 patients (68.2 ± 0.9 years). IHC was used to analyze type 9 metalloproteinase (MMP-9) and its inhibitor (TIMP-1), the role of the Ki-67 protein proliferation level and the diagnostic value of the type IV collagen distribution indicator. Results BPH is characterized by high production of MMP-9 (intensity 3 points), lack of proliferative activity of the secretory cell layer (Ki-67 less than 3.24 ± 0.61%) and degradation of collagen IV of the basement membranes, which associated with a compensatory increase in the activity of the inhibitor of matrix metalloproteinases TIMP-1 (intensity 3 points). In prostate cancer, as neoplastic changes increase, the production of the Ki-67 protein significantly increases, reflecting the proliferative activity of the secretory cell layer (from 5.3 + 1.1% to 17.2 + 9.4%). With an increase in the Gleason number over the 3rd grade, collagen fibers around the tumor cells completely disappear. The production of MMP-9 type in adenocarcinomas is significantly reduced and the destruction of collagen IV of the basement membrane of glandular structures and stroma occurs due to a progressive (up to an intensity of 0.8 + 0.4 points as anaplastic changes increase) decrease in the production of the inhibitor of matrix metalloproteinases TIMP-1 (p≤0.05). High production of MMR-9 in BPH is blocked by a high content of the TIMP-1 inhibitor. In adenocarcinomas, the production of MMP-9 is significantly reduced, but this protease destroys collagen IV of the basement membrane of glandular structures due to the absence or weak production of TIMP-1, which blocks the proteolytic effect of this enzyme on connective tissue collagen. The low production of the TIMP-1 inhibitor in adenocarcinomas can also explain the invasive properties of these tumors. Conclusions Comprehensive analysis of biomarkers in IHC can improve the diagnosis of prostate cancer and reduce the number of "unnecessary" biopsies. Disclosure No.

Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. Astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. Here, we performed an unbiased proteomic analysis and identified a fibronectin-derived peptide called Anastellin (Ana) that was unique to the Timp1KO astrocyte secretome. Ana was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Ana is known to act upon the sphingosine-1-phosphate receptor 1, and we determined that Ana also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-experimental autoimmune encephalomyelitis ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 (Timp1KO) had no effect. Analysis of Timp1 and fibronectin (FN1) transcripts from primary human astrocytes from healthy and multiple sclerosis (MS) donors revealed lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Last, analyses of proteomic databases of MS samples identified Ana peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high disease activity. A role for Ana in MS as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and innate remyelination potential in the MS brain.

Anti inflammatory and human chondrocyte protective effect of Drynariae rhizoma extract

Purpose: To evaluate the anti-inflammatory properties of Drynariae rhizome (Polypodiaceae) extract (DRE) on lipopolysaccharide RAW264.7 macrophage cells and its protective effects on interleukin (IL)-1β-stimulated SW1353 chondrocyte cells.
 Methods: The anti-inflammatory effect of DRE (12.5, 25, 50, and 100 µg/mL) were measured by 5-lipooxygenase (LO) and cyclooxygenase (COX)-2 enzyme inhibitory activities, nitric oxide (NO) production in LPS-stimulated RAW264.7 cells using Griess reagent assay. Prostaglandins (PGE2) and leukotriene (LTB4) production were measured using enzyme-linked immunosorbent assay (ELISA). To investigate the protective effect on chondrocytes, the level of matrix metalloproteinases (MMP)-3, -9, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was determined using zymography assay and ELISA kit in SW1353 cells stimulated with IL-1β.
 Results: Drynariae rhizome (Polypodiaceae) extract (DRE) had a significant (p < 0.05) inhibitory effect on 5-LO and COX-2 enzymes in a concentration-dependent manner compared to untreated group. In RAW264.7 cells, levels of NO, PGE2 and LTB4 of DRE group were significantly (p < 0.05) decreased in study group (treated with DRE) at 12.5 μg/mL compared to the group treated with LPS (100 ng/mL) alone. Additionally, in SW1353 human chondrocytes stimulated with IL-1β, MMP-3 and MMP-9 were significantly (p < 0.05) suppressed in DRE-treated group compared to IL-1β group alone, with no significant impact on TIMP-1 production.
 Conclusion: Drynariae rhizome (Polypodiaceae) extract (DRE) possesses anti-inflammatory and cartilage protection effects. It therefore has the potential to be developed as an effective natural pharmaceutical agent for inflammation or osteoarthritis.

Omeprazole induces profibrotic gene expression in rat kidney: implication of TGF-β/Smad signaling pathway

Proton pump inhibitors (PPIs) are one of the most commonly prescribed medications. However, PPI usage is linked to a higher risk of both acute and chronic renal damage by mechanisms not entirely known. The present study demonstrates that omeprazole (10 mg/kg body weight, i.p.) causes TGF-β/Smad signaling activation and subsequent expression of the profibrotic genes CTGF and TIMP-1 in rat kidney. Increased production of CTGF and TIMP-1 accompany activation of the TGF-β/Smad signaling cascade. However, simultaneous treatment of omeprazole and the TGF-β inhibitor, disitertide (P144) (1 mg/kg body weight i.p.) suppresses the TGF-β/Smad signaling pathway and subsequent production of CTGF and TIMP-1. Additionally, TGF-β level in rat kidney was highly reduced in animals treated with the ROS (reactive oxygen species) scavenger, N-acetyl cysteine (NAC) (100 mg/kg body weight i.p.) before omeprazole administration. Furthermore, the reduction in SOD activity brought by omeprazole was returned to the normal level in those animals. However, MDA level increased by omeprazole was highly reduced in the presence of NAC. Collectively, the current findings demonstrate that omeprazole has the ability to promote the expression of the profibrotic genes CTGF and TIMP-1 in a ROS and TGF-β dependent manner. The present study suggests the co-use of ROS scavenger to improve the therapeutic use of the PPI omeprazole.

Platelet-rich plasma-derived exosomes promote rotator cuff tendon-bone healing

BackgroundRotator cuff tear (RCT) is the most common type of shoulder joint injury, platelet-rich plasma-derived exosomes (PRP-exos) are highly promising in tissue repair and regeneration. The purpose of this study was to determine the function of PRP-exos in rotator cuff tendon-bone healing. MethodsPRP-exos were isolated from the rabbit whole blood by differential ultracentrifugation and characterized through transmission electron microscopy assay, nanoparticle tracking analysis, and western blotting. Alkaline phosphatase and Von Kossa staining were used to show tendon-derived stem cell (TDSC) differentiation. RT-qPCR and western blotting were performed to detect COL II, SOX-9, and TIMP-1. To determine the therapeutic effects of PRP-exos in vivo. Thirty New Zealand white rabbits were divided into control, model, and PRP-exos groups. The RCT animal model was constructed. The changes in tendon-bone tissue were determined by HE staining. Contents of COL-II, SOX-9, and TIMP-1 were determined by immunohistochemistry staining. ResultsPRP-exos were successfully isolated from rabbit blood. PRP-exos promoted TDSC proliferation and differentiation and also induced tendon-specific markers COL II, SOX-9, and TIMP-1 production. In vivo study revealed that PRP-exos promoted early healing of injured tendons. Rabbits treated with PRP-exos had better tissue arrangement in the tear site. Additionally, the contents of COL II, SOX-9, and TIMP-1 were also increased in the RCT rabbit model after PRP-exos treatment. ConclusionsPRP-exos enhanced tendon-bone healing by promoting TDSC proliferation and differentiation. This finding indicates that PRP-exos can serve as a promising strategy to treat rotator cuff tendon-bone healing.

Effects of low level laser on periodontal tissue remodeling in hPDLCs under tensile stress

This study aimed to investigate the effect of Low-Level Laser Therapy (LLLT) on human Periodontal Ligament Cells (hPDLCs) under tension stress. Primary hPDLCs were obtained using the tissue culture method, and P3 cells were utilized for the subsequent experiments. The study comprised four groups: a blank control group (Group B), a laser irradiation group (Group L), a tension stress group (Group T), and a laser + tension stress group (Group LT). Mechanical loading was applied using an in-vitro cell stress loading device at a frequency of 0.5 Hz and deformation of 2% for two hours per day for two days. Laser irradiation at 808 nm GaAlAs laser was administered 1 h after force loading. Cell samples were collected after the experiment. Bone and fiber remodeling factors were analyzed using PCR and Western blot. Flow cytometry was employed to assess the cell cycle, while ROS and Ca2+ levels were measured using a multifunctional enzyme labeling instrument. The results revealed that laser intervention under tension stress inhibited the expression of osteogenic differentiation factors, promoted the expression of osteoclast differentiation factors, and significantly increased the production of collagen factors, MMPs, and TIMPs. The LT group exhibited the most active cell cycle (P < 0.05). LLLT not only enhanced Ca2+ expression in hPDLCs under tension stress, but also stimulated the production of ROS. Overall, our findings demonstrate that LLLT effectively accelerated the proliferation of hPDLCs and the remodeling of periodontal tissue, possibly through the regulation of ROS and Ca2+ levels in hPDLCs.

Fibrogenic and fibrolytic potential of differently activated human macrophages

Macrophages are involved in the regulation of fibrogenesis and turnover of the extracellular matrix. One way to perform this function is through the production of profibrotic and fibrolytic factors including fibronectin, laminin, collagen, and extracellular matrix proteases. The production of most of them has been well studied in experimental models; however, much remains unclear regarding human macrophages. Therefore, the aim of this study was to study the content of extracellular matrix proteases (MMP-2 and MMP-9, cathepsin L), their inhibitors (TIMP-1), and collagen (type I) in supernatants of differently activated human macrophages. We compared macrophages differentiated by M-CSF or GM-CSF and further polarized in M1 with lipopolysaccharide, in M2a with IL-4, and in M2c with dexamethasone. Macrophages was obtained from peripheral blood monocytes. The content of MMPs, TIMP, cathepsin, and collagen was determined using appropriate ELISA kits. The results obtained demonstrate that differentiation factors are more important for the production of the above factors compared to polarizing stimuli (lipopolysaccharide, IL-4, dexamethasone). Moreover, macrophages differentiated by M-CSF showed predominantly antifibrotic activity because of pronounced MMPs production, while GM-CSF-induced cultures, on the contrary, were characterized by profibrotic properties due to the high level of TIMP-1 and type I collagen. M1, M2a, and M2c, induced by M-CSF, differed only in MMP-2 production, and M2a produced this metalloproteinase more than other subtypes. In the case of GM-CSF-differentiated cells, a higher level of production of TIMP-1 and, to a lesser extent, type I collagen was characteristic of M1, whereas M2c have minimal concentration of them among GM-CSF-induced macrophage subtypes. Concerning the level of cathepsin L production was relatively constant and did not depend on the generation conditions (differentiation and polarizing signals). Thus, the data obtained help to identify macrophage subtypes with anti- or profibrotic potential and may be useful for the development of cell therapy for diseases associated with fibrogenesis dysregulation.

Role of club cell 16-kDa secretory protein in asthmatic airways.

Club cell 16-kDa secretory protein (CC16) is a pneumoprotein and functions as an anti-inflammatory or antioxidant protein. However, altered levels of serum CC16 as well as their effect on airways inflammation have not been fully evaluated. We recruited 63 adult asthmatics on maintenance medications and 61 healthy controls (HCs). The asthmatic subjects were divided into two groups according to the result of bronchodilator responsiveness (BDR) test: the present BDR (n = 17) and absent BDR (n = 46) groups. Serum CC16 levels were measured by ELISA. As an in vitro study, the effect of Dermatophagoides pteronyssinus antigen 1 (Der p1) on the production of CC16 in airways epithelial cells (AECs) according to a time-dependent manner was assessed; the effects of CC16 protein on oxidative stress system, airways inflammation and remodelling were tested. Serum CC16 levels showed significantly higher in the asthmatics than in the HCs (p < .001) with a positive correlation with FEV1 % (r = .352, p = .005). The present BDR group had significantly lower levels of serum CC16, FEV1 % and MMEF%, but showed higher level of FeNO than the absent BDR group. Serum CC16 levels (below 496.0 ng/mL) could discriminate the present BDR group from the absent BDR group (area under the curve = 0.74, p = .004). In vitro testing demonstrated that Der p1 exposure significantly induced CC16 release from AECs for 1 h, which was progressively decreased after 6 h and followed by MMP-9 and TIMP-1 production. These findings were associated with oxidant/antioxidant disequilibrium and restored by CC16 treatment (but not dexamethasone). Decreased CC16 production contributes to persistent airways inflammation and lung function decline. CC16 may be a potential biomarker for asthmatics with BDR.

Interleukin-17 and Tumor Necrosis Factor Show a Functional Hierarchy to Regulate the Production of Matrix Metalloproteases by Monocytes from Patients with Psoriasis.

Interleukin-17 (IL-17) and tumor necrosis factor (TNF) regulate tissue remodeling through matrix metalloproteinases (MMPs). It is not yet clear whether these cytokines have a functional hierarchy in psoriasis. Serum levels of TNF (1,403 versus 1,058 pg/mL), IL-17 (1,528 versus 820 pg/mL), MMP-1 (1,999 versus 1,039 pg/mL), and MMP-9 (1,950 versus 1,561 pg/mL) were higher in psoriasis subjects (n = 60) than in control subjects (n = 60). Tissue inhibitor of MMPs (TIMP-1; 1,374 versus 1,218 pg/mL) was lower in psoriasis subjects. Serum IL-17 was correlated with MMP-2 (rs = 0.40) and TIMP-1 (rs = -0.26) levels. Unstimulated production of MMP-1, MMP-2, and MMP-9 by monocytes was higher in psoriasis subjects, whereas TIMP-1 production was lower. TNF stimulation increased all MMPs, whereas TIMP-1 production was unchanged. IL-17 stimulation increased all MMPs, whereas TIMP-1 production was decreased in psoriasis subjects. MMP-9 production was higher in monocytes stimulated with IL-17 compared with TNF. TIMP-1 production was decreased more by IL-17 than by TNF, but only in psoriasis cells. MMP-1/TIMP-1, MMP-2/TIMP-1, and MMP-9/TIMP-1 ratios were higher after IL-17 stimulation (compared with TNF stimulation) in psoriasis subjects; this occurred in controls only for the MMP-2/TIMP-1 ratio. IL-17 has a greater ability than TNF to dysregulate the MMPs/TIMP-1 balance, supporting IL-17 blockade as first-line treatment in cutaneous psoriasis.

TNF-induced metalloproteinase-9 production is associated with neurological manifestations in HTLV-1-infected individuals.

HTLV-1-infected individuals may develop a neurologic inflammatory condition known as HTLV-1-associated myelopathy (HAM/TSP), in which the high production of TNF is observed. These patients exhibit higher proviral loads, enhanced production of proinflammatory cytokines and lymphocyte proliferation in comparison to asymptomatic HTLV-1 carriers and those presenting overactive bladder (OAB-HTLV-infected). Metalloproteinases (MMPs) are known to degrade the components of the blood-brain barrier, favoring the migration of infected cells into the central nervous system. Moreover, the unbalanced production of MMPs and their inhibitors (TIMPs) has also been associated with tissue damage. The present work studied the production of MMP-9 and TIMPs in HTLV-1-infected individuals with and without neurological manifestations. HAM/TSP patients presented higher concentrations of MMP-9 in peripheral blood mononuclear cell (PBMC) culture supernatants, as well as a higher MMP-9/TIMP-3 ratio when compared to the other groups studied. MMP-9 levels positively correlated with proviral load and TNF in OAB-HTLV-infected individuals, and the in vitro neutralization of TNF significantly decreased MMP-9 levels in PBMC culture supernatants. Our findings indicate an association between MMP-9 production and the proinflammatory state associated with HTLV-1 infection, as well as HAM/TSP.

Staphylococcus aureus β-Toxin Exerts Anti-angiogenic Effects by Inhibiting Re-endothelialization and Neovessel Formation.

Staphylococcus aureus causes severe, life-threatening infections that often are complicated by severe local and systemic pathologies with non-healing lesions. A classic example is S. aureus infective endocarditis (IE), where the secreted hemolysin β-toxin potentiates the disease via its sphingomyelinase and biofilm ligase activities. Although these activities dysregulate human aortic endothelial cell activation, β-toxin effect on endothelial cell function in wound healing has not been addressed. With the use of the ex vivo rabbit aortic ring model, we provide evidence that β-toxin prevents branching microvessel formation, highlighting its ability to interfere with tissue re-vascularization and vascular repair. We show that β-toxin specifically targets both human aortic endothelial cell proliferation and cell migration and inhibits human umbilical vein endothelial cell rearrangement into capillary-like networks in vitro. Proteome arrays specific for angiogenesis-related molecules provided evidence that β-toxin promotes an inhibitory profile in endothelial cell monolayers, specifically targeting production of TIMP-1, TIMP-4, and IGFBP-3 to counter the effect of a pro-angiogenic environment. Dysregulation in the production of these molecules is known to result in sprouting defects (including deficient cell proliferation, migration, and survival), vessel instability and/or vascular regression. When endothelial cells are grown under re-endothelialization/wound healing conditions, β-toxin decreases the pro-angiogenic molecule MMP-8 and increases the anti-angiogenic molecule endostatin. Altogether, the data indicate that β-toxin is an anti-angiogenic virulence factor and highlight a mechanism where β-toxin exacerbates S. aureus invasive infections by interfering with tissue re-vascularization and vascular repair.

Interleukin-1β More Than Mechanical Loading Induces a Degenerative Phenotype in Human Annulus Fibrosus Cells, Partially Impaired by Anti-Proteolytic Activity of Mesenchymal Stem Cell Secretome.

Mesenchymal stem/stromal cell (MSC)–based therapies for low back pain and intervertebral disc (IVD) degeneration have been emerging, despite the poor knowledge of their full mechanism of action. As failure of the annulus fibrosus (AF) is often associated with IVD herniation and inflammation, the objective of the present study was to investigate the impact of the MSC secretome on human AF cells exposed to mechanical loading and a pro-inflammatory environment. Human AF cells isolated from IVD biopsies from patients with adolescent idiopathic scoliosis (AIS) or disc degeneration (DD) were exposed to physiological cyclic tensile strain (CTS) for 72 h in a custom-made device, with or without interleukin (IL)-1β medium supplementation. AF cells stimulated with CTS + IL-1β were then treated with secretome from IL-1β–preconditioned MSCs for 48 h. AF cell metabolic activity, gene expression, protein secretion, matrix metalloproteinase (MMP) activity, and tissue inhibitor of MMPs (TIMP) concentration were evaluated. Expanded AF cells from AIS and DD patients revealed similar metabolic activity and gene expression profiles. CTS stimulation upregulated collagen type I (COL1A1) expression, while IL-1β significantly stimulated IL-6, IL-8, MMP-1, and MMP-3 gene expression and prostaglandin E2 production by AF cells but downregulated COL1A1. The combination of CTS + IL-1β had a similar outcome as IL-1β alone, accompanied by a significant upregulation of elastin. The MSC secretome did not show any immunomodulatory effect on CTS + IL-1β–stimulated AF cells but significantly decreased MMP-1, MMP-2, MMP-3, and MMP-9, while increasing the production of TIMP-1. The obtained results demonstrate a stronger impact of the inflammatory milieu on human AF cells than upper physiologic mechanical stress. In addition, a new MSC mechanism of action in degenerated IVD consisting of the modulation of AF MMP activity was also evidenced, contributing to the advancement of knowledge in AF tissue metabolism.

BFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling

IntroductionBasic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration. ObjectivesThe purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling. MethodsSCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining. ResultsSCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05). ConclusionbFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.

Cigarette Smoke Extract Stimulates MMP-2 Production in Nasal Fibroblasts via ROS/PI3K, Akt, and NF-κB Signaling Pathways.

Cigarette smoke exposure has been shown to be associated with chronic rhinosinusitis and tissue remodeling. The present study aimed to investigate the effects of cigarette smoke extract (CSE) on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) production in nasal fibroblasts and to determine the underlying molecular mechanisms. Primary nasal fibroblasts from six patients were isolated and cultured. After the exposure of fibroblasts to CSE, the expression levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by real-time PCR, ELISA, and immunofluorescence staining. The enzymatic activities of MMP-2 and MMP-9 were measured by gelatin zymography. Reactive oxygen species (ROS) production was analyzed using dichloro-dihydro-fluorescein diacetate and Amplex Red assays. PI3K/Akt phosphorylation and NF-κB activation were determined by Western blotting and luciferase assay. CSE significantly increased MMP-2 expression and inhibited TIMP-2 expression but did not affect MMP-9 and TIMP-1 expression. Furthermore, CSE significantly induced ROS production. However, treatment with ROS scavengers, specific PI3K/Akt inhibitors, NF-κB inhibitor, and glucocorticosteroids significantly decreased MMP-2 expression and increased TIMP-2 expression. Our results suggest that steroids inhibit CSE-regulated MMP-2 and TIMP-2 production and activation through the ROS/ PI3K, Akt, and NF-κB signaling pathways in nasal fibroblasts. CSE may contribute to the pathogenesis of chronic rhinosinusitis by regulating MMP-2 and TIMP-2 expression.

Production of factors involved into fibrosis regulation by various types of human macrophages

Macrophages (Mφ) play a key role in regulation of fibrogenesis, including proliferation of fibroblasts and myofibroblasts, differentiation of progenitor cells into myofibroblasts, as well as synthesis and secretion of the extracellular matrix, mainly collagen. The direction of the Mφ effects (stimulation or suppression) is determined by a number of factors, including the stage of the fibrotic process and the Mφ functional phenotype dependent on the signals of microenvironment. One of the feasible ways of the fibrogenesis regulating is the secretion of pro- or antifibrotic factors such as matrix metalloproteinases, inhibitors of metalloproteinases and some cytokines. However, existing data on ability to secrete these factors by various subpopulations of human Mφ are rare and controversial. The aim of this study was to characterize the ability of human M1, M2a, and M2c Mφ differentiating in the presence of GM-CSF to produce matrix metalloproteinases (MMP-9) and their tissue inhibitors (TIMP-1), as well as some cytokines and growth factors. As compared to M2 macrophages, the M1 macrophages polarized by lipopolysaccharide produced significantly more TNFα, IL-6 and IL-2 that have pro-inflammatory activity and are able to initiate a fibrotic process. In turn, M2a Mφ stimulated by IL-4 were characterized by a high level of VEGF production and, at the same time, low levels of TNFα and IL-6, which may determine the important role of these cells at the proliferative stage of fibrosis and stimulation of extracellular matrix deposition. Finally, M2c Mφ polarized by dexamethasone, exhibited the М2а-like cytokine profile, i.e., VEGF was actively produced against the background of low TNFα and IL-6 synthesis. Moreover, all three Mφ subpopulations did actively secrete MMP-9 and TIMP-1, without significant difference in production of these factors. However, M2c Mφ differed by a significantly higher MMP-9/TIMP-1 ratio index compared to M1 and M2a Mφ, and it is crucial at the rearrangement stage of the fibrotic process. Thus, the production of MMP-9 and TIMP-1, together with other pleiotropic cytokines and growth factors by various Mφ subtypes may reflect their role in regulation of fibrotic process at various stages.

Cancer Burden Is Controlled by Mural Cell-β3-Integrin Regulated Crosstalk with Tumor Cells

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-β3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-β3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell β3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by β3-integrin, providing a previously unrecognized mechanism of cancer growth control.

Alisma Shugan Decoction (ASD) Ameliorates Hepatotoxicity and Associated Liver Dysfunction by Inhibiting Oxidative Stress and p65/Nrf2/JunD Signaling Dysregulation In Vivo.

BackgroundLiver fibrosis, defined as the aberrant accumulation of extracellular matrix (ECM) proteins such as collagen in the liver, is a common feature of chronic liver disease, and often culminates in portal hypertension, liver cirrhosis, and hepatic failure. Though therapeutically manageable, fibrosis is not always successfully treated by conventional antifibrotic agents. While the traditional Chinese medicine (TCM) Alisma Shugan Decoction (ASD) has several health benefits, including anti-inflammation, anti-oxidation, and limitation of cardiovascular and respiratory disorders, it remains unclear if it has any hepato-protective potential.Material/MethodsThe present study examined the therapeutic effect of ASD in thioacetamide (TAA)-induced liver injury and fibrosis rat models.ResultsWe demonstrated that 50 mg/kg ASD significantly reversed TAA-induced elevation of alanine or aspartate transaminase levels, elicited no dyscrasia, and conferred a 40% (p<0.01) or 20% (p<0.05) survival advantage, compared to rats treated with TAA or TAA+ASD, respectively. Treatment with ASD reversed TAA-induced liver injury and fibrogenesis via repression of α-SMA protein and reduction of the collagen area and fibrosis score. Concurrently, ASD markedly suppressed the mRNA expression of fibrogenic procollagen, ICAM-1, MMP2, MMP9, and MMP13, and production of TIMP-1, ICAM-1, CXCL7, or CD62L cytokine in rat liver injury models. Interestingly, ASD-elicited reduction of liver injury and fibrogenesis was mediated by dysregulated p65/NrF-2/JunD signaling, with a resultant 3.18-fold (p<0.05) increase in GSH/GSSH ratio, and a 3.61-fold (p<0.01) or 1.51-fold (p<0.01) reduction in the 4-hydroxynonenal and malondialdehyde (MDA) levels, respectively, indicating reduced oxidative stress in the ASD-treated rats, and suggesting an hepato-protective role for ASD.ConclusionsIn conclusion, the present study provides supplementary evidence of the therapeutic benefit of ASD as an efficient treatment option in cases of liver injury and fibrosis. Further large-cohort validation of these findings is warranted.

Histological response and expression of collagen, metalloproteinases MMP-1 and MMP-9 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 in fetal membranes following open intrauterine surgery: an experimental study

Objective To characterize aspects of the repair process by evaluating the tissue collagen density, metalloproteinases and tissue inhibitor of matrix metalloproteinases in the fetal membranes following open fetal surgery for myelomeningocele (MMC). Design Experimental. Setting Two Brazilian hospitals in 2013–2014. Population 30 fetal membranes collected after elective cesarean deliveries, in patients who underwent open fetal surgery for MMC intrauterine repair. Methods Regions within and surrounding the scar area and regions distant from the surgical site were evaluated for collagen concentration and expression of MMP-1, MMP-9, TIMP-1 and TIMP-2. Results Collagen was increased in regions of scar formation (14.4 ± 2.7%) as compared to unaffected regions (8.0 ± 1.9%) (p < .001). The mean score of MMP-9 in the area of both the scar and suture was also increased above that observed in normal regions (p < .05). Conversely, MMP-1 was reduced in the scar when compared to the normal region and the area adjacent to the scar (suture region) (p < .05). TIMP-1 was increased in the suture region compared to the normal region (p < .05) while TIMP-2 was reduced in the scar region when compared to the other two regions (p < .05). The membrane repair process was also influenced by the number of previous pregnancies and gestational age at the time of surgery. Conclusion Reparative activity of the fetal membrane after open fetal surgery involves up-regulation of collagen production and differential involvement of MMPs and TIMPs.

Galectin-1-Related Modulation of Trophoblast Endothelial Interactions by Integrins α1 and β1.

During normal trophoblast invasion, integrins α6β4 are downregulated, and α1β1 are upregulated in invasive cytotrophoblast cells. In preeclampsia both interstitial and endovascular invasion are shallow and cytotrophoblasts fail to upregulate α1β1 and downregulate α6β4. This study aims to investigate the role of integrins α1β1 and α6β4 on cellular pathways influencing trophoblast integration into endothelial cellular networks in vitro. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells pre-incubated with 20μg/ml of neutralizing antibodies (anti-α1, β1, α6, β4, α1+β1, or α6+β4) for 1h were then co-cultured with endothelial networks with the neutralizing antibodies for 24h. Fluorescent images were captured, and quantified utilizing Image J. Cells were retrieved to analyze mRNA expression of galectin-1, TIMP-1, and PAI-1 by quantitative PCR. MMP-2, MMP-9, free sFlt-1, and PlGF from conditioned media were measured by ELISA. The integration of trophoblast cells into endothelial cellular networks was inhibited by anti-β1(- 28± 3%, p< 0.0001), and increased by anti-α6(+ 19± 5%, p< 0.01). Galectin-1 mRNA expression was decreased by anti-α1(- 35± 7%, p< 0.001), anti-β1(-23± 5%, p< 0.05), and anti-α1+β1(- 35± 5%, p< 0.001). The mRNA expression of TIMP-1 was inhibited by anti-α1(- 59 ± 9%, p< 0.01) and anti-β1(- 63± 7%, p< 0.001) while PAI-1 mRNA expression was increased by anti-α1 + β1(+ 285 ± 70%, p< 0.0001). In the conditioned medium, anti-α1 reduced MMP-2(-28± 1%, p< 0.001), MMP-9(-27± 8%, p< 0.01), and sFlt-1(-27± 5%, p< 0.001) production. Anti-β1 reduced MMP-2(- 15± 2%, p< 0.05) production. There were no changes in PlGF. Appropriate integrins α1β1 modulate trophoblast cell integration into endothelial cellular networks in vitro through invasive pathways including galectin-1, TIMP-1, PAI-1, MMP-2, and MMP-9 production.

Effect of sulfated galactans from red seaweed Gracilaria fisheri on extracellular matrix production in human dermal fibroblast

Fibroblasts are derived from mesenchymal stem cells that play an important role in produce the extracellular matrix (ECM). ECM creates a tissue microenvironment for regulation of cell signaling. The loss of ECM plasticity is associated with several pathologies, especially those involving chronic inflammation, therefore, the ECM represents a potential therapeutic target for certain conditions. Sulfated galactans was extracted from red seaweed Gracilaria fisheri that previous studies have been reported about its activities on anti‐oxidant, anti‐tumour, anti‐melanogenesis, and anti‐bacteria. Therefore, the aim of this study was to investigate the effect of potential of sulfated galactans from red seaweed G. fisheri on enhancing the ECM production. Cytotoxicity was tested following determination of ECM‐related gene and protein expression on human dermal fibroblast (HDF) by quantitative RT‐PCR and enzyme‐linked immunosorbent assay (ELISA), respectively. The result showed that sulfated galactans not effects to survival rate of HDF. Additionally, we found that sulfated galactans was able to modulate ECM production by regulating the collagen, elastin, hyaluronic acid, MMP‐1, and TIMP production. The present results demonstrating the effects of a natural multi‐component compound on ECM composition, highlighted the possibility of pharmacologically modulating ECM molecules. The recovery and the maintenance of ECM homeostasis might be considered as a potential therapeutic goal to ameliorate pathological conditions.Support or Funding InformationThis work was financially supported by Navamindradhiraj University Research Fund.