TIMP-1 Protein Research Articles

Highly-expressed micoRNA-21 in adipose derived stem cell exosomes can enhance the migration and proliferation of the HaCaT cells by increasing the MMP-9 expression through the PI3K/AKT pathway

ObjectiveWound healing remains a challenge in burns and trauma fields. Adipose derived stem cells exosomes (AD-exos) had been confirmed to have a positive effect on the wound healing and the migration and proliferation of keratinocyte. However, the mechanism of the AD-exos is still unclear. The objective of this article is to observe the function of the miR-21 expressed in the adipose AD-exos and the effect on migration and proliferation of the HaCaT cells. Materials and methodsThe full layer dermal wound of BALb/c mouse was used to observe the vitro effect of the AD-exos and detect the expression of miR-21.The co-culture systems were established by transwell plates for observing the migration, proliferation, apoptosis rate, detecting the RNA, and protein expression in different treated groups. MiR-21 plasmid was used to over-express miR-21 by transfection of HaCaT cells. GW4869 was used to inhibit the secreting of exosomes from ADSCs. ResultsThe results showed that both ADSCs and the AD-exos could improve the wound healing process of BALb/c mouse full layer skin wound at a similar level, especially at the 7th day post surgery when compared to the control group (p < 0.01) and the highly expressed miR-21 was detected (p < 0.01 compared with control group and p < 0.001 compared to other microRNAs) in the treated groups at the same time point. AD-exos could obviously enhance the migration and proliferation of the HaCaT cells (p < 0.01), and fell back to the same level when the exosomes inhibitor--GW4869 was added compared with control group (p > 0.05). Over-expressed miR-21 could also significantly improve the migration and proliferation of HaCaT cells. But both AD-exos and miR-21 had no significantly effect on the apoptosis rate of HaCaT cells (p > 0.05 compared with each other). Over-expression of miR-21 plasmid could decrease the TGF-βI protein level (p < 0.001 vs. control group) in HaCaT cells while TGF-βI protein level increased again when antagomiR-21 was added in (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 expression of HaCaT cells could be increased by the transfect ion of miR-21 plasmid (p < 0.001 vs. empty plasmid group) and decreased by antagomiR-21 (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 appeared to have influence on MMP-9 and TIMP-2 (p < 0.001 compared to control group and p < 0.001 compared to TGF-βI group) but not MMP-2 and TIMP-1 (p > 0.05 compared to control group and TGF-βI group). These processes might act through PI3K/AKT pathway. ConclusionThis research provide the experimental evidence that the miR-21 is highly expressed in AD-exos and can significantly accelerate the wound healing process and enhance the migration and proliferation of the HaCaT cells. Over-expressed miR-21 can inhibit the TGF-βI expression and excess TGF-βI can also have negative feedback influence on miR-21. We have found a reliable evidence that these two factors can act on HaCaT cells by influencing MMP-2 and TIMP-1 protein expression through the PI3K/AKT signal pathway. These results may provide a potential perspectives on improving the wound healing.

Puerarin enhance vascular proliferation and halt apoptosis in thiram-induced avian tibial dyschondroplasia by regulating HIF-1α, TIMP-3 and BCL-2 expressions.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate pesticide used for crop protection and storage. But, it's widespread utilization is associated with deleterious growth plate cartilage disorder in broilers termed as avian tibial dyschondroplasia (TD). TD results in non-mineralized and less vascularized proximal tibial growth plate cartilage causing lameness and poor growth performance. This study investigated the therapeutic potential of puerarin against thiram toxicity in TD affected chickens. One-day-old broiler chickens (n=240) were alienated into three equal groups i.e. control, TD and puerarin (n=80) and were offered standard feed. Additionally, TD and puerarin groups were offered thiram at 50mg/kg of feed from 4 to 7 days for TD induction followed by puerarin therapy at 120mg/kg to puerarin group only from 8 to 18 days for TD treatment. Thiram feeding to TD and puerarin group chickens caused lameness, mortality, and increased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels and growth plate (GP) size and upregulated HIF-1α expression. Besides, the production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and the expressions of TIMP-3 and BCL-2 were decreased (p<0.05). Puerarin alleviated lameness, enhanced angiogenesis and growth performance and serum and antioxidant enzymes, decreased apoptosis and recuperated GP width by significantly downregulating HIF-1α and upregulating the TIMP-3 and BCL-2 mRNA and protein expressions in puerarin group chickens (p<0.05). In conclusion, the toxic effects associated with thiram can be mitigated using puerarin.

Biological and Proteomic Characteristics of an Immortalized Human Pancreatic Stellate Cell Line.

Human pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during chronic pancreatitis (CP). However, primary PSCs have a short lifespan in vitro, which seriously affects their use in various applications. We have established a stable immortalized human PSC line (HP-1) by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated human PSCs. HP-1 cells express cytoskeleton proteins including glial fibrillary acidic protein (GFAP), α-smooth muscle actin (α-SMA), vimentin and desmin, and are typical of PSCs, which are high transfeciability and viable in 0.5% serum. The cells express receptors such as TGFβR2, PDGFR, TGF-β pseudoreceptor Bambi and PPRPγ that are commonly found in PSCs. HP-1 cells are similar to activated human PSCs in that they have enhanced expression of α-SMA, CTGF, Col1 and TIMP-2 mRNAs or proteins, as well as decreased expression of MMP-1/2 mRNAs or proteins in response to TGF-β1 stimulation. Comparative proteomics revealed 4,537 shared proteins between HP-1 cells and PSCs and no single protein in HP-1 cells versus PSCs. Statistical analysis reveals no significantly difference between HP-1 cells and PSCs in their expression of proteins associated with matrix and matrix remodeling. The similarity between HP-1 cell and PSC is further shown by the finding that only 9 proteins are differentially up-regulated > ± 2-fold in HP-1 cells and 13 proteins are up-regulated > ± 2-fold in PSCs and none of these proteins include ECM proteins, cytokines, growth factors or matrix remodeling regulatory proteins. Therefore, HP-1 cells can be used as an effective tool for the study of PSC-mediated pancreatic fibrosis.

Metformin Inhibits Proliferation and Tumor Growth of QGP-1 Pancreatic Neuroendocrine Tumor Cells by Inducing Cell Cycle Arrest and Apoptosis.

Pancreatic neuroendocrine tumors (pNETs) are rare pancreatic neoplasms, and therapeutic options for pNETs are limited. Metformin is an anti-hypoglycemic drug that appears to have anticancer effects. However, little is known about the effect of metformin on pNETs. In this study, we investigated the anti-proliferative effect of metformin on a human pNET cell line. The anti-proliferative properties of metformin were evaluated in QGP-1 and NCI-H727 cells using a cell counting kit-8 assay. Xenograft mouse models were used to assess the tumor effect in vivo. Metformin inhibited the proliferation and anti-tumor growth of QGP-1 cells, accompanied by their arrest during the cell cycle at the G0/G1 phase. Immunohistochemical analysis of tumor tissues revealed down-regulation of cyclin D1 and proliferating cell nuclear antigen in the metformin-treated group. Additionally, metformin induced apoptosis, and the expression of survivin and claspin were decreased in metformin-treated QGP-1 cells according to the apoptosis array. Furthermore, the angiogenic related protein TIMP-1 was down-regulated, and its miRNA expression was altered by metformin in QGP-1 cells. Taken together, our study demonstrated the therapeutic potential of metformin and provides molecular mechanistic insights into its anti-tumoral effect on pNETs. This study is the first one describing anti-tumoral effects in pNETs.

Tissue inhibitor of metalloproteinase proteins inhibit teratoma growth in mice transplanted with pluripotent stem cells.

Pluripotent stem cells (PSCs) can serve as an unlimited cell source for transplantation therapies for treating various devastating diseases, such as cardiovascular diseases, diabetes, and Parkinson's disease. However, PSC transplantation has some associated risks, including teratoma formation from the remaining undifferentiated PSCs. Thus, for successful clinical application, it is essential to ablate the proliferative PSCs before or after transplantation. In this study, neural stem cell-derived conditioned medium (NSC-CM) inhibited the proliferation of PSCs and PSC-derived neural precursor (NP) cells without influencing the potential of PSC-NP cells to differentiate into neurons in vitro and prevented teratoma growth in vivo. Moreover, we found that the NSC-CM remarkably decreased the expression levels of Oct4 and cyclin D1 that Oct4 directly binds to and increased the cleaved-caspase 3-positive cell death through the DNA damage response in PSCs and PSC-NPs. Interestingly, we found that NSCs distinctly secreted the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 proteins. These proteins suppressed not only the proliferation of PSCs in cell culture but also teratoma growth in mice transplanted with PSCs through inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 activity. Taken together, these results suggest that the TIMP proteins may improve the efficacy and safety of the PSC-based transplantation therapy.

Orexin-A Exerts Equivocal Role in Atherosclerosis Process Depending on the Duration of Exposure: In Vitro Study.

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure.

Follistatin Mitigates Myofibroblast Differentiation and Collagen Synthesis of Fibroblasts from Scar Tissue around Injured Flexor Tendons.

PurposeThe aim of this study was to investigate the effect of FST gene on the inhibition of fibrosis in fibroblastic cells from scar tissue around repaired zone II flexor tendons.Materials and MethodsImmunohistochemistry was conducted on fibroblast cells transfected with adenovirus-LacZ (Ad-LacZ) as a marker gene (control), or with adenovirus-FST (Ad-FST) as a therapeutic gene. Fibroblast cultures without adenoviral exposure served as controls.ResultsFibroblastic cells transfected with Ad-FST demonstrated significant decrease in collagen type I, MMP-1, MMP2, and α-SMA mRNA expressions compared to those transfected with Ad-LacZ. In addition, fibroblastic cells transfected with Ad-FST exhibited significant decrease in MMP-1, TIMP-1, fibronectin, PAI-1, TRPV4, α-SMA, desmin, and PAX7 protein expressions.ConclusionBased on these findings, we conclude that FST may be a novel therapeutic strategy for preventing scar adhesions around repaired tendons by inhibiting fibroblasts from differentiating into myofibroblasts, in addition to producing type I collagen and regulating extracellular matrix turnover via the downregulation of MMP-1 and TIMP-1. FST may also decrease contracture of the scar by inhibiting Ca2+-dependent cell contraction.

Staphylococcus aureus on the effect of expression of MMPs/TIMPs and uPA system in bovine mammary fibroblasts.

Bovine mammary fibrosis is characteristic of chronic in injury in response to diverse pathogens. Staphylococcus aureus (S.aureus) is a frequent cause of mastitis in bovine and is prone to persistent infection. Diverse studies have shown MMPs/TIMPs and uPA system as a potent target for the treatment of fibrosis. However, pathogenesis of S. aureus-induced mammary fibrosis has not been completely defined. BMFBs treated with heat-inactivated S. aureus (105, 106, and 108CFU/mL) for 6, 12, 24, and 48h. Total RNA and protein were isolated from the treatments and controls of BMFBs samples. MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, COL Ⅰ, uPA, uPAR and PAI-1 gene and protein expression were examined by RT-qPCR and Western blot analysis. Gelatin zymography assay was performed to assess the levels of MMP-2 and MMP-9 enzyme secreted. BMFBs treated with heat-inactivated S. aureus increased mRNA and protein expression levels of MMP-1, MMP-2, MMP-3, MMP-9 and MMP-13, and heat-inactivated S. aureus induced TIMP-1, TIMP-2 and COL Ⅰ expression. There was a clear activation of MMP-2 in the presence of heat-inactivated S. aureus in the conditioned medium from the BMFBs, whereas MMP-9 was no significantly altered. Moreover, uPA system was activated in BMFBs to S. aureus. Activation of the uPA system together with its impact on the MMPs levels could play a significant role in S. aureus-induced BMFBs with mechanism of ECM metabolism, MMPs/TIMPs and uPA system could participate in bovine mammary fibrosis.

Regulatorg Mchanism of MiR-152 on Proliferation, Metastasis and Tumorigenicity of SHI-1 Cells

To investigate the regulatory mechanism of miR-152 on proliferation, metastasis and tumorigenesis of human acute monocytic leukemia SHI-1 cells. The purchased SHI-1 cell line was treated with miR-152 over-expression (miR-152 agomir group) or miR-152 inhibition (miR-152 antagomir group), and the negative control (NC) group was set up. The cell proliferation of each group was detected by CCK-8 assay. Scratch healing assay was employed to determine the migration of cells. Transwell assay was used to measure the invasion of cells. The expressions of Cyclin D1, Caspase-3, MMP-2, TIMP-2, E-cadherin and N-cadherin were detected by Western blot. The flow cyronetry with annexin V-FITC/PI double staining was applied to detect the cell apoptosis. The tumorigenesis of cells was examined by tumor formation experiment in nude mice. Compared with the NC group, the cell proliferation, migration and invasion ability in miR-152 agomir group were significantly decreased (P＜0.05), while that in miR-152 antagomir were significantly up-regulated (P＜0.05) . Compared with the NC group, the protein expression of Cyclin D1, MMP-2, N-cadherin were down-regulated in miR-152 agomir group, but the protein expression of Caspase-3, TIMP-2 and E-cadherin were all up-regulated siginificantly. At the same time, the apoptosis were enhanced, but the timorigenicity in nude mice were significantly decreased (all P＜0.05). The protein expression of Cyclin D1, MMP-2, N-cadherin in miR-152 antagomir group, showed high levels but Caspase-3, TIMP-2 and E-cadherin protein showed low levels in comparison with NC group. At the same time, the apoptosis was decreased but the timorigenicity in nude mice was significantly enhanced (all P＜0.05) . miR-152 can inhibit the proliferation, metastasis and tumorigenesis of SHI-1 cell line, at the same time induce cell apoptosis, thus providing a theoretical basis for the treatment of acute lymphoblastic leukemia.

Evaluation of metalloproteinases-2, -9, and -13 post photobiomodulation in mice talocrural joint.

The extracellular matrix (ECM) is the main constituent of connective tissue with structural and regulatory functions, stimulating cell differentiation and proliferation. Moreover, ECM is a dynamic structure in the constant remodeling process, which is controlled by a balance between metalloproteinases (MMPs) and their inhibitors (TIMPs). Photobiomodulation (PBM) is widely described in the literature and applied in clinical practices, although its effects on ECM have not yet been elucidated. Therefore, it was evaluated if PBM could alter ECM components, such as MMP-2, -9, -13, and TIMP-2 from mice talocrural joints. Mice were divided into 3 groups (n = 6): control, PBM 3Jcm-2, and PBM 30Jcm-2. A low-level laser (830nm, 10mW, 0.05 irradiated area, energy densities 3Jcm-2 and 30Jcm-2, the irradiation time of 15 and 150s, respectively, continuous wave) was applied on the joint for 4 consecutive days. mRNA levels of metalloproteinases genes (MMP-2, MMP-9, and MMP-13), their regulator (TIMP-2), and protein expressions of MMP-13 and TIMP-2 were quantified. PBM can alter only mRNA relative levels of MMP-2 at 30Jcm-2 (p < 0.05), while MMP-9, MMP-13, and TIMP-2 mRNA relative levels did not demonstrate statistical differences for any of the groups (p > 0.05). Regarding protein expressions, MMP-13 demonstrated positive-labeled cells, only in articular cartilage, although the cell quantification did not demonstrate statistical differences when compared with the control group (p > 0.05). TIMP-2 did not present positive-labeled cells for any tissues evaluated. Our results indicate that PBM can alter MMP-2 mRNA relative level but cannot alter MMP-9, MMP-13, and TIMP mRNA relative levels. Moreover, both MMP-13 and TIMP-2 proteins were also unaltered after PBM.

Neural stem cells and the secreted proteins TIMPs ameliorate UVB-induced skin photodamage

UV-induced skin damage is involved in ROS overproduction and the overexpression of matrix metalloproteinases (MMPs), which are inhibited by TIMPs (tissue inhibitor of neural stem cells (NSCs)). These proteins may be associated with skin regeneration through the activation of TIMP proteins, but there have been no reports of treatment of skin photodamage using NSCs and their secreted proteins TIMP-1 and TIMP-2. Here we investigated the photoprotective role of NSCs and their TIMP proteins for the inhibition of UVB-irradiation damage in fibroblasts in SKH-1 mice. SKH-1 hairless mice were divided into three groups (n = 4 per group): normal, treatment, and control groups. The latter two groups were dorsally exposed to UVB irradiation for 12 weeks. After UVB irradiation, treatments with NSC-CM and its secreted factors TIMP-1 and TIMP-2, markedly ameliorated the photodamage triggered by the increase in MMP expression and activity through ROS production, and the subsequent activation of the NF-κB pathway in UVB-irradiated fibroblasts and the treatment mouse group. In addition, the topical application of NSC-CM to mice in the treatment group after irradiation clearly inhibited the expression of γ-H2AX, a DNA damage marker, through the activation of the DNA repair enzyme Rad50. These results demonstrate that NSC-CM or TIMPs proteins can ameliorate skin photodamage induced by UVB-irradiation in in vitro and in vivo systems.

Spatial analysis of RECK, MT1-MMP, and TIMP-2 proteins during early Xenopus laevis development

Proper cell-cell and cell-ECM interactions are vital for cell migration and patterning of the vertebrate embryo. MMPs and their inhibitors, RECK and TIMPs, are all differentially expressed during embryogenesis to regulate such ECM remodeling and cell interactions. MT1-MMP, RECK, and TIMP-2 are unique amongst other ECM-regulating proteins as they act in the pericellular space. Past studies have shown that RECK and TIMP-2 interact with MT1-MMP on the cell surface, thereby influencing cell behaviour as well as the microenvironment immediately surrounding the cells. We investigated the localization of RECK, TIMP-2, and MT1-MMP proteins throughout early X. laevis development using immunohistochemistry. We found that during neural tube formation, axis elongation, and organogenesis, RECK, TIMP-2, and MT1-MMP proteins show highly similar localization patterns, particularly in the ectoderm and in the dorsal-ventral differentiation of the neural tube. Our data suggests they function together during patterning events in early Xenopus development.

Influence of clodronate and compressive force on IL-1ß-stimulated human periodontal ligament fibroblasts.

The aim of this study was to investigate in vitro the effect of clodronate on interleukin-1ß (IL-1ß)-stimulated human periodontal ligament fibroblasts (HPdLFs) with the focus on inflammatory factors of orthodontic tooth movement with and without compressive force. HPdLFs were incubated with 5μM clodronate and 10ng/mL IL-1ß. After 48h, cells were exposed to 3h of compressive force using a centrifuge. The gene expression of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), matrix metalloproteinase 8 (MMP-8), and the tissue inhibitor of MMP (TIMP-1) was analyzed using RT-PCR. Prostaglandin E2 (PGE-2), IL-6, and TIMP-1 protein syntheses were quantified via ELISA. Compressive force and IL-1ß induced an overexpression of COX-2 gene expression (61.8-fold; p < 0.05 compared with control), diminished by clodronate (41.1-fold; p < 0.05 compared with control). Clodronate slowed down the compression and IL-1ß induced IL-6 gene expression (161-fold vs. 85.6-fold; p < 0.05 compared with control). TNF-α was only slightly affected without statistical significance. Clodronate reduced IL-1ß-stimulated MMP-8 expression with and without compressive force. TIMP-1 on gene and protein level was downregulated in all groups. Analyzing the MMP-8/TIMP-1 ratio, the highest ratio was detected in IL-1ß-stimulated HPdLFs with compressive force (21.2-fold; p < 0.05 compared with control). Clodronate diminished IL-1ß-induced upregulation of MMP-8/TIMP-1 ratio with (11.5-fold; p < 0.05 compared with control) and without (12.5-fold; p < 0.05 compared with control) compressive force. Our study demonstrates a slightly anti-inflammatory effect by clodronate under compressive force in vitro. Additionally, the periodontal remodeling presented by the MMP-8/TIMP-1 ratio seems to be diminished by clodronate. Reduction of pro-inflammatory factors and reduction of periodontal remodeling might explain reduced orthodontic tooth movement under clodronate intake.

Disequilibrium in MMPs and the tissue inhibitor of metalloproteinases in different segments of the varicose great saphenous vein wall.

Varicose great saphenous veins (VGSVs) are a common disorder with a high incidence, but the pathogenesis is unclear. This study was designed to measure the changes in matrix metalloproteinases (MMPs) and the tissue inhibitor of metalloproteinases (TIMPs) in different segments from VGSV walls to determine the relationship between MMPs, TIMPs expression, and expansion of the venous wall. Twenty-one VGSV and 12 normal great saphenous vein (GSV) specimens were collected. Venous walls in the two groups, expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 proteins, protein-positive expression ratios, mRNA expression, and protein content were determined by immunohistochemistry, PCR, and western blot. The MMP-2, MMP-9, TIMP-1, and TIMP-2 protein-positive expression ratios, mRNA expression in the upper, middle, and lower segments in the VGSV group were significantly higher than the corresponding regions in the GSV group, respectively. The MMP-2, MMP-9, TIMP-1, and TIMP-2 protein-positive expression ratios, mRNA expression, and protein concentrations in the lower segments in the VGSV group were also significantly higher than the upper and middle segments in the VGSV group and the corresponding regions in the GSV group, respectively. Under high hemodynamics, disequilibrium of MMPs and TIMPs from VGSVs exists within the upper, middle, and lower segments of VGSVs. These results suggested that MMPs and TIMPs participate in the process of venous wall remodeling and may be one of the mechanisms associated with the formation and development in varicose veins.

Expression of long-stranded noncoding RNA HOXA transcript at the distal tip in bladder cancer and its effect on proliferation, migration and invasion of bladder cancer cells

Objective To investigate the expression of long-stranded non-coding RNA HOXA transcript at the distal tip (HOTTIP) (lncRNA HOPTTIP) in bladder cancer cells (BCC) and its effect on proliferation, migration and invasion of BCC and potential mechanisms. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression differences of HOTTIP between normal tissue and bladder cancer tissues, normal cell and bladder cancer cell. Cell counting kit-8 (CCK-8) was used to observe the proliferation levels of BCC. Cell cycle progression of BCC was detected by flow cytometry. Cell invasion and metastasis were observed by transwell and wound healing experiments. Western blotting assay was used to detect the protein expressions of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMPs). Results The expression level of HOTTIP in BCC (1.943±0.251) was significantly higher than that of paracancerous tissue (1.002±0.091), and the difference was statistically significant (P<0.05); The expression of HOTTIP in human normal epithelial cells (SV-HUC-1) was significantly lower than that of 5637 and T24 (1.003±0.092 vs. 2.241±0.306 and 1.692±0.203), with significant difference (P<0.05). After si-HOTTIP and si-NC intervention 48h on 5637 and T24 cells, the proliferation of 5637 (0.881±0.182 vs. 1.824±0.193) and T24 (0.623±0.094 vs. 1.332±0.153) cells decreased significantly (P<0.05), the cell cycle were stuck into G0/G1 phase, rate of invasion and migration were reduced, MMP-2 and MMP-9 protein expression levels decreased, TIMP1 and TIMP2 protein expression levels increased. Conclusion Long-stranded non-coding RNA (LncRNA) HOTTIP is down-regulated in bladder cancer tissues and cells, and promotes the proliferation, migration and invasion of BCC by decreasing MMPs and increasing TIMPs. Key words: Long non-coding RNAHOXA transcript at the distal tip; Bladder cancer; Proliferation; Migration; Invasion

Exercise Increases Myocardial TIMP‐2 Protein Levels and Is Associated with Cardioprotection against Myocardial Ischemia‐Reperfusion Injury

Coronary artery disease (CAD) remains the leading cause of death worldwide. The most common manifestation of CAD is myocardial infarction (MI), a result of ischemia and/or reperfusion (IR) injury of the myocardium. It is well established that exercise training improves myocardial tolerance to IR, a phenomenon known as exercise‐induced cardioprotection, but the cellular adaptations resulting in cardioprotection against IR injury are not well understood. Recent work has linked IR injury to the activation of the matrix metalloproteinase‐2 (MMP‐2). MMPs are known to be involved in the degradation of the extracellular matrix, and play a key role in scar formation following MI. While this extracellular role of MMP‐2 in MI has been well established, recent studies suggest an additional, intracellular, role of MMP activation in IR injury. MMP‐2 has been shown to have intracellular proteolytic targets in cardiomyocytes, which could contribute to the cell death seen following IR. Tissue inhibitor of metalloproteinases (TIMPs) are inhibitors of the MMPs, and TIMPs have been found in cardiomyocytes. It is thus possible that exercise provides cardioprotection against IR injury through 1) decreasing MMP‐2 protein levels, or 2) inhibiting MMP‐activation by increasing the protein levels of the TIMPs. However, no study to date has investigated the effect of exercise training on the protein levels of MMP‐2 and TIMP‐2 following IR injury. Therefore, the specific aims of this study were to determine the effects of exercise training on MMP‐2 and TIMP‐2 protein expression, and to identify regional differences in the levels of these proteins. To address these questions, we studied 3‐month old Sprague‐Dawley rats randomly assigned to sedentary (SED) or exercise (EX) groups (n=10/group). EX rats were run on a treadmill for 60 min/day, 5 days/week, for 8 weeks. IR was induced by occluding the LAD for 45 min, followed by 24 hr of reperfusion. Hearts were excised and sectioned and stained to delineate the zone at risk (ZAR) from the unaffected area (UA). Infarct size was expressed as a percentage of the ZAR. Hearts from EX rats exhibited smaller infarcts compared to SED rats (21.4% vs. 37.2%; P &lt; 0.05). MMP‐2 and TIMP‐2 levels in ventricular homogenates were determined by Western blot. MMP‐2 protein content was increased in the ZAR compared to the UA in both EX and SED rats (P &lt; 0.05). There was no difference in MMP‐2 protein levels between EX or SED rats. TIMP‐2 protein levels were increased in the hearts from EX rats compared to SED rats (P &lt; 0.05). There was no difference in TIMP‐2 levels between the ZAR and UA in EX or SED rats. These results show that 1) MMP‐2 levels were significantly higher in the ZAR compared to the UA, suggesting an intracellular role of MMP‐2 in IR injury, and 2) exercise training leads to an increase in myocardial TIMP‐2 protein levels, suggesting elevated TIMP‐2 inhibition of MMP‐2 as a potential mechanism of exercise‐induced cardioprotection.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Status of MMP 9 and TIMP 1 in Pregnant women with Preeclampsia in Indian Population

IntroductionMatrix metalloproteinases (MMPs) specifically MMP 9 is a key regulator of vascular and uterine spiral artery remodelling and its activity is controlled at multiple levels, including gene transcription, activation of its latent forms and endogenous inhibition by tissue inhibitors of metalloproteinases [TIMPs (specifically TIMP 1)]. Alteration in MMP 9 and TIMP 1 expression may contribute to uterine and vascular dysfunction leading to adverse pregnancy outcomes such as Preeclampsia (PE) and FGR. Therefore, in the present study, we sought to analyse the levels of this enzyme (MMP 9) and its regulator (TIMP 1) in the patients of PE.MethodsThe blood samples from the enrolled pregnant women [Total: 60, Preeclamptic: 30, Normotensive, Non‐proteinuric (Controls): 30)] were used for determining the mRNA expression of MMP 9 and TIMP 1 using qRT‐PCR. The sera of the above subjects were used for measuring MMP 9 and TIMP 1 protein levels using commercially available ELISA kits (R and D system). However, 30 caesarean delivered placentae (15 PE and 15 normotensive) were used to analyse the mRNA expression of MMP 9 and TIMP 1 using qRT‐PCR. Also, the MMP 9 and TIMP1 protein expression was seen by immunohistochemistry (IHC) and immunofluorescence (IF) staining. Zymography was performed for MMP 9 characterization and TIMP 1 expression was evaluated using western blot in these placentae.ResultsMMP 9 mRNA expression reduced in both plasma (2.66 folds) and placentae (6.66 folds) of PE patients as compared to controls. However, TIMP 1 mRNA expression was elevated in both plasma (2.23 folds) and placentae (8.32 folds) of PE patients as compared to controls. ELISA results demonstrated decrease in MMP 9 (p=0.0526; Wilcoxon matched‐pairs signed rank test) and increase in TIMP 1 (p=0.9133; Wilcoxon matched‐pairs signed rank test) protein levels in PE patients as compared to healthy controls. Immunolocalisation revealed stronger expression of MMP 9 in placental syncytiotrophoblasts and stroma in control placentae as compared to PE placentae. However, TIMP 1 intensity reduced in placental villi and decidual cells in the placentae from normotensive non‐proteinuric pregnant women. MMP 9 activity (zymography) was also found to be less and TIMP‐1 (western blot) upregulated in PE placentae as compared to control placentae.ConclusionUnderstanding the role of MMP 9 and TIMP 1 in uteroplacental and vascular remodelling and function could help design new approaches for prediction and management of preeclampsia.Ethical Clearance: Ref. No. IECPG‐101/21.03.2018Support or Funding InformationF.No.IR‐516/2018/RSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MT1-MMP evaluation in neointimal hyperplasia in the late follow-up after prosthesis implantation.

Vascular surgical interventions are often burdened with late complications, including thrombosis or restenosis. The latter is generally caused by neointimal hyperplasia. Although extracellular matrix (ECM) remodelling is an important part of neointima formation, this process is not clearly understood. The aim of the study was to assess the content and activity of membrane-type 1 matrix metalloproteinase in human neointima in the late stages of its development. Matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 were also evaluated. The research was performed on neointima samples collected during secondary vascular interventions from patients with chronic limb ischaemia who developed vascular occlusion at 6-18 months after aorto/ilio-femoral bypass grafting. The control material consisted of segments of femoral arteries collected from organ donors. Western blot and/or ELISA were used for the determination of MT1-MMP and TIMP-2 expression. The activity of MT1-MMP was measured by fluorometric assay and that of MMP-2 by zymography. We demonstrated significantly increased MT1-MMP protein content in neointima when compared to normal arteries. However, the activity of MT1-MMP was significantly lower in neointima than in control samples. The decreased MT1-MMP activity was concomitant with reduced activity of MMP-2. The TIMP-2 protein levels in neointima and normal arteries were not significantly different. The results of our study suggest that the reduced activity of MT1-MMP and consequently MMP-2 in human neointima may play a role in decreased degradation of ECM components and thus promote neointimal overgrowth.

Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.

Green tea-induced epigenetic reactivation of tissue inhibitor of matrix metalloproteinase-3 suppresses prostate cancer progression through histone-modifying enzymes.

Green tea polyphenols (GTPs) and their major constituent, epigallocatechin-3-gallate (EGCG), have been reported to demonstrate many interesting biological activities, including anticancer properties. Recent studies on prostate cancer provide strong evidence that epigenetic mechanisms are major players in the regulation of matrix metalloproteinases (MMPs) and their binding partner tissue inhibitor of MMPs (TIMPs) involved in prostate cancer progression. Here we demonstrate that GTP/EGCG mediate epigenetic reactivation of TIMP-3 that plays a key role in suppressing invasiveness and cancer progression. Treatment of human prostate cancer DUPRO and LNCaP cells with 10 µg/mL GTP and 20 µM EGCG induced TIMP-3 mRNA and protein expression. This transcriptional activation of TIMP-3 was associated with the decrease in the expression of both enhancers of zeste homolog 2 (EZH2) and its catalytic product trimethylation of histone H3 at lysine 27 (H3K27me3) repressive marks at the TIMP-3 promoter with an accompanying increase in histone H3K9/18 acetylation. In addition, GTP/EGCG treatment significantly reduced class I histone deacetylase (HDAC) activity/expression and EZH2 and H3K27me3 levels in prostate cancer cells. EGCG/GTP exposure also reduced MMP-2/MMP-9 gelatinolytic activity and abrogated invasion and migration capabilities in these cells. Silencing of EZH2 and class I HDACs strikingly increased the expression of TIMP-3 independent of DNA methylation. Furthermore, clinical trials performed on patients undergoing prostatectomy consuming 800 mg EGCG (Polyphenon E) up to 6 weeks and grade-matched controls demonstrate an increase in plasma TIMP-3 levels. A marked reduction in class I HDACs activity/expression and EZH2 and H3K27me3 levels were noted in GTP-supplemented prostate tissue. Our findings highlight that TIMP-3 induction, as a key epigenetic event modulated by green tea in restoring the MMP:TIMP balance suppresses prostate cancer progression.